【作者】 蔡爱珍；

【导师】 陈凛；

【作者基本信息】 中国人民解放军军医进修学院 ， 普外科， 2010， 博士

【摘要】 目的探讨乙酰肝素酶(heparanase, HPSE)基因对胃癌细胞生物学行为的影响；阐明HPSE基因在胃癌细胞信号转导、生长发育、炎症反应及肿瘤转移等多项生物过程中的可能分子机制方法1 siRNA对胃癌细胞乙酰肝素酶基因表达及其生物学行为的影响①应用RT-PCR技术筛选HPSE基因高表达的胃癌细胞株。②设计、合成反义寡脱氧核苷酸(AS-ODN)及对照寡脱氧核苷酸(NS-ODN),转染高表达HPSE基因的胃癌细胞株,利用Real-Time PCR技术确定沉默效果最佳的siRNA浓度,并运用Real-Time PCR和Western Blot技术分别在mRNA和蛋白水平验证对HPSE基因表达的沉默效果。③应用透射电镜观察HPSE siRNA对胃癌细胞SGC-7901形态学的影响；采用Annexin V-FITC/PI双染流式细胞术检测HPSE siRNA对胃癌细胞SGC-7901凋亡的影响。④应用Matrigel侵袭实验和细胞迁移实验研究HPSE siRNA对胃癌细胞SGC-7901侵袭力和迁移能力的影响。2乙酰肝素酶反义寡核苷酸对胃癌细胞基因表达谱的影响应用Affymetrix Human U133 Plus2基因芯片技术,分别提取siRNA抑制HPSE基因表达前后胃癌细胞SGC-7901的总RNA,反转录成cDNA,进一步荧光标记后进行芯片杂交,杂交结果经扫描及软件分析,最后Ratio值为Cy3/Cy5(即实验组／对照组)。差异基因筛选标准为正标Ratio(Cy3/Cy5)≥2,同时反标Ratio(Cy3/Cy5)≤0.5.分析siRNA抑制HPSE基因表达后,胃癌细胞中受HPSE调控的下游基因表达谱的变化,并且部分上调及下调基因的表达水平用Real-Time PCR进行了验证。对于差异表达在2倍以上的基因,我们利用博奥公司的MAS3.0分析软件进行了GO分析,并对差异表达在1.5倍以上的基因进行了Pathway分析。结果1 siRNA对胃癌细胞乙酰肝素酶基因表达及其生物学行为的影响①RT-PCR技术检测胃癌细胞株中HPSE基因表达情况,结果显示SGC-7901细胞表达HPSE最高,故选用SGC-7901细胞为实验细胞模型。②RT-PCR技术及Real-Time PCR实验证实沉默效果最好的siRNA浓度为10 nM。③Real-Time PCR及Western Blot检测：相对于阴性对照NC组(无义siRNA对照组),siRNA沉默组的HPSE基因的mRNA水平和蛋白表达量显著降低。④siRNA沉默HPSE基因后,透射电镜结果显示胃癌细胞SGC-7901形态学发生典型的凋亡现象；Annexin V-FITC/PI双染流式细胞术检测发现凋亡细胞明显增多,由正常细胞的1.58%升高为8.45%。⑤Transwell细胞侵袭实验结果显示,siRNA沉默HPSE基因后的胃癌细胞SGC-7901侵袭到Transwell膜后面的细胞数较阴性对照组显著减少(P<0.01),表明HPSE基因沉默明显降低胃癌细胞的侵袭能力。利用细胞划痕实验检测细胞的迁移能力,结果显示,HPSE基因沉默的胃癌细胞在划痕后24h、36h的宽度明显大于对照组(P<0.05),表明抑制HPSE基因可明显降低胃癌细胞的迁移能力。2乙酰肝素酶反义寡核苷酸对胃癌细胞基因表达谱的影响Affymetrix芯片结果表明,siRNA沉默HPSE基因后,胃癌细胞SGC7901中VEGF信号通路的中心介导分子PRKCA和PRKCB的表达明显下降；介导VEGFR-2通路的锚定蛋白SHB、VEGFR-2的相互作用蛋白VE-cadherin、炎症因子IL-1a／IL-1b表达下降,介导天然免疫的分子DEFB1、黏附分子CD44表达升高。结论1 siRNA沉默HPSE基因后,降低胃癌细胞SGC-7901的侵袭和迁移能力；2 siRNA沉默HPSE基因表达后,胃癌细胞SGC-7901中VEGF信号通路的中心介导分子PRKCA和PRKCB的及传递该通路信号的重要分子SHB和VE-cadherin表达均下降,但跨膜糖蛋白CD44表达升高,这些分子改变可能共同参与了HPSE干涉后的血管生成抑制过程。3 siRNA沉默HPSE基因后,SHB、VE-cadherin表达下降,CD44表达升高,FAK通路受到抑制,这些因素可能共同作用导致胃癌细胞SGC-7901的凋亡增加。4 siRNA沉默HPSE基因后,炎症因子IL-1a／IL-1b表达下降、能抑制炎症反应的CD44黏附分子表达增加以及炎症负调控因子HO-1表达增加,可能共同参与HPSE干涉后对炎症反应的抑制作用。更多还原

【Abstract】 Object:To investigate the impact of heparanase(HPSE) gene on gastric cancer biological behaviors and clarify the underlying molecular mechanisms of HPSE gene in many biological processes of the gastric cancer cells such as signal transduction, tumor angiogenesis, inflammation and tumor metastasis.Methods:1 Effects of HPSE siRNA on HPSE gene expression and biological behaviors of gastric cancer cells①RT-PCR was performed to screen gastric cancer cell lines with high HPSE gene expression.②Both of antisense oligodeoxyribonucleotide (AS-ODN) and control oligodeoxyribonucleotide (NS-ODN) targeting HPSE were designed and chemically synthesized. The screened gastric cancer cells were transfected with optical concentration of AS-ODN and NS-ODN tested by Real-Time PCR, and expression of HPSE at mRNA and protein levels were examined by Real-Time PCR and Western Blot in order to verify the silencing effects.③Effects of silencing HPSE on the morphology of gastric cancer cell SGC-7901 were observed by transmission electron microscope (TEM). The role of silencing HPSE on apoptosis of gastric cancer cell was detected by Annexin V-FITC/PI flow cytometry.④Effects of silencing HPSE on the abilities of invasiveness and migration of gastric cancer cell SGC-7901 were tested by Matrigel invasion assay and cell migration assay.2 Effects of HPSE antisense oligonucleotides on gene expression profiling of gastric cancer cellsAffymetrix Human U133 Plus2 gene chips were used to identify the gene expression changes after HPSE expression knockdown. Total RNA extracted from gastric cancer cells with AS-ODN and NS-ODN was reversely transcripted into cDNA to make chip cross-fertilization after fluorescent labeling. The cross-fertilization results were scanned and analyzed by software and then the Ratio was recorded as Cy3/Cy5 (case group/control group). The screening standards of differential gene were that positive Ratio(Cy3/Cy5)≥2 and negative Ratio (Cy3/Cy5)≤0.5. Some up and down regulated genes were verified by Real-Time PCR after HPSE expression was silenced and gene expression profiling changes of the HPSE-mediated downstream in gastric cancer cell were analyzed. Genes of differential expression that exceeded 2 folds were analyzed by "GO" tool using MAS3.0 software of CapitalBio Corporation, whereas genes that exceeded 1.5 folds were analyzed by "Pathway" tool.Results:1 Effects of HPSE silencing on HPSE gene expression and biological behavior of gastric cancer cells①RT-PCR detection showed that SGC-7901 expressed the highest HPSE levels among three gastric cancer cell lines, that’s why SGC-7901 was selected as experiment cell model.②The optimal concentration of siRNA, verified by RT-PCR and Real-Time PCR assay, was lOnM.③Real-Time PCR and Western Blot results showed that expression of HPSE at mRNA and proteins level after siRNA decreased significantly as compared with negative control group.④TEM observation showed that HPSE silenced SGC-7901 exhibited typical apoptosis cell morphology. Annexin V/PI flow cytometry analysis showed that apoptosis was increased remarkably from 1.58% in mock group to 8.45% in HPSE silenced group.⑤Transwell assay indicated that the number of cell migrated through membrane in HPSE silenced group were more less than that of mock group (P<0.01), suggesting that inhibition of HPSE expression could abolish the invasive capabilities of SGC-7901 cells. Scrape migration assay showed that the wounded empty space in HPSE silenced group was obviously wider than that of control group at 24h and 36h after being scraped (P<0.05), indicating that inhibition of HPSE could decrease the migration ability of gastric cancer cells obviously.2 Effects of silencing HPSE on down stream gene expression of gastric cancer cellsAffymetrix analysis showed that expression of PRKCA and PRKCB, central mediator molecule of VEGF signal pathway, was decreased significantly in HPSE silenced group compared with mock group. Meanwhile, the expression of SHB, an adaptor protein of VEGFR-2 pathway, and VE-cadherin which interacted with VEGFR-2, were decreased in HPSE silenced group. It is also founded the inflammatory factor IL-la/IL-lb decreased. However, expressions of innate immunity-mediating molecule DEFB1 and adhesion molecule CD44 increased when HPSE were inhibited.Conclusions:1 Silencing HPSE abolished the abilities of invasion and migration of SGC-7901;2 Expressions of PRKCA and PRKCB, central bridge molecule of VEGF signal cascades, and that of SHB and VE-cadherin, both of which were important member of this pathway, were decreased in HPSE silenced group. Meanwhile, the expression of transmembrane glycoprotein CD44 was increased. These changes might play an important role in the decreased angiogenesis when HPSE expression was attenuated;3 Gene Chip assay showed that decreased expressions of SHB and VE-cadherin, increased expression of CD44, and attenuated FAK pathway when HPSE expression was silenced. All of these might be responsible for increasing cell apoptosis after HPSE was silenced;4 Gene Chip assay also exhibited that decreased expression of inflammatory factor IL-1a/IL-1b, increased expression of anti-inflammation adhesion molecule CD44 and negative inflammation regulatory factor HO-1 in HPSE silenced group relative to mock group. All of these might be involved in the inhibition of inflammatory reaction after HPSE was silenced.更多还原