摘要
本文主要分为两大部分:
第一部分:应用基因芯片研究与子宫内膜异位症的相关基因分别取处在增生期、分泌期的两子宫内膜异位症患者的在位子宫内膜为实验对象,与非子宫内膜异位症者的在位子宫内膜作比较,应用基因芯片分析两组各自的差异基因。其中两基因芯片中共同上调或下调的基因是更有意义的。共同上调的基因有白细胞介素-6基因,Chromosome: 7; Location: 7p21 MIM: 147620 GeneID: 3569。α2-HS糖蛋白基因,Chromosome: 3; Location: 3q27 MIM: 138680 GeneID: 197。
第二部分:
1、(1)采用病例对照研究的方法,以98例子宫内膜异位症患者与99名健康成年女性外周血白细胞为样本,应用聚合酶链反应-限制性片段长度多态性技术检测两组的白细胞介素-6基因启动子-174、-572、-597位点多态性,比较两组各自的基因型和等位基因的分布频率。
(2)将如上实验中疾病组中病例分级,对比各级中白细胞介素-6基因启动子-572位点的多态性分布。
2、(1)采用病例对照研究的方法,同上实验对象,应用聚合酶链反应-限制性片段长度多态性技术检测两组的多态性,比较两组各自的α2-HS糖蛋白(AHSG)基因第七个外显子中Sac I位点基因型和等位基因的分布频率。
(2)将如上实验中疾病组中病例分级,对比各级中AHSG基因位点的多态性分布。
3、进一步研究白细胞介素-6基因启动子-572位点多态性与AHSG基因第七个外显子中Sac I位点基因多态性在子宫内膜异位症发病中的协同作用。
1, Screening related genes of morbidity of endometriosis by using cDNA chips
Objective To compare the differential expression genes between normal endometria of patients with endometriosis and child-bearing age women without endometriosis and screen related genes of morbidity of endometriosis.
Methods There were four women objects in this experiment. Two of them were patients of endometriosis. The other two were normal controls. The two patients were in different period of menstrual cycle. Their laparoscopic diagnosis was the II stage of endometriosis .One patient was in proliferation the other was in secretion. One of the two normal controls was patient of hysteromyoma the other was patient of tumor of the ovary. They were treated by laparoscopic operation and evacuated endometriosis. One normal contral was in proliferation the other was in secretion. Their normal endometria were taken out to be used. Con-RNA of all endometria were gotten out by TRIzol kit. The separation of oxybenzene and chloroform were used to depurate the RNA. After prehybridization method of Schena were used to mark explorer of cDNA through reverse transcription. cDNA of patients' endometria was marked by Cy3-dCTP and that of normal controls' endometria was marked by Cy5-dCTP.cDNA chips which contained 8096 sites were used to compare the two kinds of endometria. After allomixis and washing, the cDNA chips were scaned. Ratios of Cy3/Cy5 were analyzed by scanner. Twocondions were needed in this analysis. One condition was the signals of Cy3 and Cy5 exceeded 200 or one of the two exceeded 800, the other condition was the ratio of Cy3/Cy5 exceeded 2 or was less than 0.5. The gene witch corresponded the two conditions was gene of differential expression. The genes whose tendency of differential expression in the experimental result was sieved .The genes whose tendency of proliferation and secretion was consistent were especially needed to be pay attention to. Results 12.27% (994/8096) genes were much different in expression level in endometria were found in the two objects who were in proliferation. 6.69% (542/8096) of them were upregulated genes. 5.58% (452/8096) were downregulated. 10.28% (832/8096) genes were much different in expression level in endometria were found in the two objects who were in endometria. 5.08% (411/8096) of them were upregulated genes. 5.20% (421/8096) were downregulated . 9 genes were screened out both in the period of proliferation and secretion. Among them, only 2 genes were up-regulated, 7 genes were down -regulated. The genes of differential expression in proliferation were cDNA FLJ30195 fis,clone BRACE2001374, tissue inhibitor of metalloproteinade, hypothetical protein FLJ22116, 602628972F1 Homo sapies cDNA, 5' end, hypothetical protein FLJ11848, inhbin beta A, zinc finger homeobox lb, glutsthione peroxidase 3, core promoter element binding protein, Homo sapiens intercellular adhesion molecule I , nucleolar protein ANKT, Secretory leukoCyte protease inhibitor, interleukin 1 receptor antagonist and KRT13 etc. The genes of differential expression in secretion were Cytokine induced protein 29 kDa, 602670971F1 Homo sapies cDNA,5' end, similar to zinc finger protein 136, hemoglobin, beta, plasminogen activator, tissue, facilitated glucose transporter, member 1, bridging integrator 3, chromosome 8 open reading frame 4, secreted phosphoprotein 1,insulin-like growth factor binding protein 3mRNA for KIAA1868 protein, partial cds, th78b06.xl Homo sapiens cDNA 3'end, alpha-2A, receptor, Cytokine induced protein 29 kDa, 602670971F1 Homo sapies cDNA,5' end and similar to zinc finger protein 136 etc. The genes whose tendency of proliferation and secretion was consistent were Homo sapiens interleukin 12 rexeptor, Homo sapiens mRNA for MEMD protein, Homo sapiens integrin, alpha 6, Homo sapiens CD9 antigen, homo sapiens 5' nucleotidase, Homo sapines RAB2,member RAS oncogenefamily, Homo sapiens tumor necrosis factor supetfamily, member 13, Homo sapiens interleukin 6 and Homo sapiens alpha-2HS- glycoprotein. Conclusion cDNA chips is a technique of sequencing and quantitative analysis of nucleotide sequence. Parallel analysis of considerable genes is able to be done with only a little chip. It can detect and screen a lot of genes at the same time. It is efficient while its required volume of sample is very less than any other technique. It can analyze disease which was caused by multiple factors. It fits for the search of finding causes of endometriosis. Microarray analysis of gene expression technique is an effectual way in screening out the differentially expressed genes between patients and normal controls. Further analysis of these obtained genes will be hopeful to find the new diagnostic target about endometriosis.
2、Association of polymorphism in Interleukin-6(IL-6) gene promoter region with morbidity of endometriosis
Objective To detect the distribution of the site -174G/C, the site -572C/G and the site -597G/A in interleukin-6 gene promoter region in patients with endometriosis and normal controls. Methods Interleukin-6 gene promoter region polymorphism was detected by polymerase chain reaction- restriction fragment length polymorphism in patients with endometriosis (n=98) and normal controls (n=99).In patients with endometriosis, there were 22 patients of stage II ,35 of stage III while 41 of stagelV. Comparisons were done in patients of stage II ,III or IV when significant difference was seen in genotype, frequency or allele frequency in disease group and control grouq. Results To the site -174,the frequencies of GG,GC and CC genotypes were 100%,0% and 0% in patients with endometriosis, while 98.99%,1.01% and 0% in normal controls. No significant difference was seen in the distribution frequency of interleukin-6 genotype between patients with endometriosis and normal controls. To the site -572 the frequencies of CC,CG and GG genotypes were 55.10%, 42.86% and 2.04% in patients with endometriosis , while 70.71 %, 28.28% and 1.01% in normal controls. A significant difference was seen in two groups. To the site -597 of IL-6 gene no GA and AA genotypes were found in two groups. To the site -572 the frequencies of CC,CG and GG genotypes were59.09%, 40.90% and 0% in patients with endometriosis of stage II, 54.29%, 42.86% and 2.85% of stage III while53.66%, 43.90% and 2.44% of stage IV. No significant difference was seen in three groups. Conclusion Mutation of the sites -174 and -597 of interleukin-6 gene promoter region were rare. Its distribution frequency has nothing to do with morbilty of endometriosis. While to the site -572, its distribution frequency of genotype has something to do with morbilty of endometriosis but has nothing to do with the extent of endometriosis.
3、Distribution of interleukin-6 promoter region and AHSG gene polymorphism in patients with endometriosis
Objective To detect the distribution of polymorphism at Sac I site G/C in the seventh extron of AHSG gene and IL-6 promoter region -572 C\G in patients with endometriosis and normal controls. Methods Interleukin-6 gene promoter region and Sac I site in the seventh extron of AHSG gene polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism in patients with endometriosis(n=98) and normal controls(n=99). In patients with endometriosis, there were 22 patients of stage II ,35 of stage III while 41. of stageIV. Comparisons were done in patients of stage II,III or IV when significant difference was seen in genotype frequency or allele frequency in disease group and control grouq.
results: To IL-6 promoter region -572 the frequencies of CC,CG and GG genotypes were 55.10% , 42.86% and 2.04% in patients with endometriosis, while 70.71%,28.28% and 1.01% in normal controls. A significant difference was seen in the distribution frequency in the two groups; To Sac I site G/C in the seventh extron of AHSG gene the frequencies of GG,GC and CC genotypes were32.65%, 57.14% and 10.20% in patients with endometriosis, while 74.75%,23.23% and 2.02% in normal controls. A significant difference was seen in the distribution frequency in the two groups. To the site -572 the frequencies of CC,CG and GG genotypes were59.09%, 40.90% and 0% in patients with endometriosis of stage II, 54.29%, 42.86% and 2.85% of stage III while53.66%, 43.90% and 2.44% of stage IV. No significant difference was seen in three groups. To Sac I site G/C in the seventh extron of AHSG frequencies of GG,CG and CC genotypes were31.82%, 59.09% and 9.09% in patients with endometriosis of stage II, 31.43%, 57.14% and 11.43% of stage III while34.14%, 56.10% and 9.76% of stage IV. No significant difference was seen in three groups.
Conclusion The distribution frequency of interleukin-6 genotype and AHSG gene have something to do with morbility of endometriosis but have nothing to do with the extent of endometriosis; Genotypes C/C in IL-6 gene and G/G in AHSG gene may have a protective effect against endometriosis; There may exist some interaction between polymorphisms of IL-6 gene and AHSG gene in the pathogenesis of endometriosis.
引文
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