【作者】 唐智；

【导师】 袁贤瑞；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：研究Wnt-1诱导分泌蛋白2(WISP-2)基因在人脑星形细胞瘤中的表达情况及其与人脑星形细胞瘤发生、发展的关系。方法：采用逆转录聚合酶链反应(RT-PCR)检测47例人脑星形细胞肿瘤、10例正常脑组织中WISP-2的mRNA表达水平,并结合临床资料分析WISP-2表达水平与星形细胞瘤临床指标的相关性关系。结果：RT-PCR检测结果显示正常脑组织中无WISP-2 mRNA表达,在星形细胞瘤中相对表达量为0.677±0.445,与正常组比较差异有显著性(t=4.783,P＜0.01)。高级别组(Ⅲ,Ⅳ)与低级别组(Ⅰ,Ⅱ)WISP-2 mRNA相对表达量分别为0.924±0.438、0.478±0.344,两者比较差异有显著性(t=3.909,P<0.01),Spearman相关性分析显示WISP-2 mRNA表达水平和肿瘤病理级别具有显著性正相关关系(r=0.448,P=0.002)。WISP-2 mRNA的表达与年龄、性别、肿瘤部位无显著性相关关系(均P>0.05),与肿瘤大小有显著性关系(P<0.05)。结论：WISP-2 mRNA在人脑星形细胞瘤中有表达,其表达水平与人脑星形细胞瘤的恶性程度密切相关；提示WISP-2在人脑星形细胞瘤的发生发展过程中发挥重要的调控作用。WISP-2基因有可能作为评价人脑星形细胞瘤恶性程度的指标之一。目的：研究WSIP-2蛋白在人脑星形细胞瘤中的表达情况及其与各临床病理指标的关系。方法：采用免疫组织化学SP法检测54例人脑星形细胞瘤和10例正常脑组织中的WSIP-2蛋白表达,分析其表达水平与患者各临床病理指标之间的关系。结果：免疫组化检测结果显示正常脑组织细胞中无WISP-2蛋白阳性表达(0／10)。WISP-2蛋白在星形细胞瘤组织内呈阳性表达,阳性率为64.8%(35／54),与正常脑组织中表达比较有显著性意义(P<0.05)。高级别组(Ⅲ,Ⅳ)与低级别组(Ⅰ,Ⅱ>中WISP-2蛋白表达阳性率分别为80.8%(21／26)、50.0%(14／28)，两者比较差异有显著性(P<0.05)。WISP-2蛋白的表达与年龄、性别、肿瘤部位无显著性关系(均P>0.05),与肿瘤大小有显著关系(P<0.05)。通过Spearman相关分析表明,WISP-2蛋白表达水平随着肿瘤的级别升高而增高(r=0.370,P=0.006),两者呈正相关性关系。结论：WSIP-2蛋白在人脑星形细胞瘤中高表达,与人脑星形细胞瘤的病理分级有正相关性,有可能作为星形细胞瘤恶性程度的指标。目的：构建沉默人WISP-2基因表达的siRNA序列并筛选出有效siRNA序列,为体外研究WISP-2的生物学功能提供材料。方法：通过RT-PCR检测WISP-2 mRNA在U251细胞株的表达,设计5个靶向WISP-2的RNA干扰序列的小干扰RNA,通过脂质体转染U251细胞,RT-PCR及Western blot筛选有效干扰序列,荧光倒置显微镜观察转染效果,最终得到人WISP-2基因沉默的U251细胞株。结果：WISP-2 mRNA在U251细胞株中表达,组5(WISP-2-289 siRNA)沉默WISP-2表达后,RT-PCR及Western blot方法检测到WISP-2 mRNA和蛋白表达水平分别为0.113±0.111, (19.03±1.44)%,与其组1(空白对照组)、组2(阴性对照组)、组3(WISP-2-639 siRNA)、组4(WISP-2-307 siRNA)比较,差异有显著性意义(P<0.05)。荧光倒置显微镜观察转染效率为(87.03±11.56)%。结论：筛选出合适的WISP-2 siRNA干扰序列,为进一步体外研究WISP-2基因在U251细胞株中的作用提供基础.。目的：探讨siRNA沉默WISP-2基因表达对U251细胞生物学特性的影响,以明确其在星形细胞瘤肿瘤形成过程中可能的作用及机制。方法：实验分3组：空白对照组(未作任何干扰的U251细胞株)；阴性对照组(加入非特异性siRNA/Lipofectamine复合物的U251细胞株)；siRNA干扰组(为WISP-2-siRNA-289-脂质体转染的U251细胞株)。通过MTT法、ranswell法、流式细胞仪PI染色法检测人WISP-2基因表达对U251细胞的增殖、迁移、凋亡能力的影响,并检测Bcl-2、Bax蛋白在细胞凋亡过程中的表达。结果：MTT检测发现在相同的时间点,siRNA干扰组组的OD值明显高于空白对照组和阴性对照组(P<0.05)；检测迁移能力实验中siRNA干扰组肿瘤细胞的运动迁移能力明显下降,穿过聚碳酸酯膜的细胞数量较对照组明显下降,与对照组相比有明显区别(P<0.05)；培养48h进行流式细胞仪检测可见,siRNA干扰组平均凋亡率为(16.59±1.40)%,空白组平均凋亡率为(3.13±0.34)%,阴性组平均凋亡率为(3.42±0.48)%,siRNA干扰组与阴性对照组、空白对照组比较差异有统计学意义(P<0.01)。在检测增殖、迁移、凋亡实验中,阴性对照组和空白对照组无明显差异(P>0.05)。siRNA干扰组WISP-2蛋白表达水平为(18.67±1.40)%明显低于空白对照组(70.18±1.82)%和阴性对照组(69.41±1.77)%,且差异有统计学意义(P<0.01),两对照组WISP-2蛋白表达水平比较差异无显著意义(P＞0.05),siRNA干扰组Bcl-2蛋白表达水平为(29.67±1.47)%,明显低于空白对照组(49.51±1.71)%和阴性对照组(49.07±1.35)%,且差异有统计学意义(P<0.01),两对照组Bcl-2蛋白表达水平比较差异无显著意义(P＞0.05)。siRNA干扰组Bax蛋白表达水平为(31.62±1.32)%,明显低于空白对照组(23.98±1.47)%和阴性对照组(24.06±1.55)%,且差异有统计学意义(P<0.01),两对照组Bax蛋白水平比较差异无显著意义(P＞0.05)。Spearman相关性分析显示,siRNA沉默WISP-2表达后,WISP-2蛋白的表达与Bcl-2蛋白的表达具有正相关性(r=0.995,P<0.05),即前者表达越低,后者表达也越低；siRNA沉默WISP-2表达后,WISP-2蛋白的表达与Bax蛋白的表达具有负相关性(r=-0.944,P<0.05),即前者表达越低,后者表达越高。结论：在U251细胞中,siRNA沉默WISP-2基因表达后,可抑制U251细胞的增殖、侵袭能力,增加U251细胞凋亡。并提示通过抑制WISP-2蛋白表达后,下调Bcl-2/Bax蛋白表达的比值来诱导U251细胞凋亡。目的：探讨联合应用WISP-2 siRNA和足叶乙甙(VP16)对U251细胞药物敏感性的影响并探讨其作用机制。方法：应用转染试齐LipofectaminTM2000将WISP-2 siRNA转入U251细胞,同时加入VP16联合培养,48h后予MTT法检测U251细胞增殖、流式细胞仪检测U251细胞凋亡变化。结果：足叶乙甙(VP16)在浓度为1μg/mL就可以表现出对U251细胞的抑制作用,U251细胞随着药物作用时间的延长和药物浓度的增加而明显抑制,具有明显的量效和时效依赖性。MTT法显示空白对照组、阴性对照组、siRNA干扰组、VP16组和siRNA干扰+VP16组OD值分别为0.257±0.011、0.252±0.015、0.166±0.011、0.155±0.012、0.076±0.014,siRNA干扰组和VP16组细胞生长速度较空白对照组和阴性对照组显著降低(P<0.05),siRNA干扰+VP16组生长速度较siRNA干扰组和VP16组显著降低(P<0.05),空白对照组与阴性对照组比较无统计学差异(P＞0.05)。联合应用WISP-2 siRNA和足叶乙甙(VP16)能明显降低U251细胞增殖能力,WISP-2基因沉默能增加U251细胞对VP16增殖抑制的敏感性。流式细胞仪检测细胞凋亡结果显示空白组平均凋亡率为(3.06±0.39)%,阴性组平均凋亡率为(3.28±0.57)%,siRNA干扰组平均凋亡率为(15.72±1.55)%,VP16干预组平均凋亡率为(14.76±1.41)%,siRNA干扰+VP16组平均凋亡率为(33.09±2.13)%,统计分析结果显示siRNA干扰组和VP16组与阴性对照组、空白对照组比较凋亡增加,差异有统计学意义(P<0.01),siRNA干扰+VP16组细胞凋亡率较siRNA干扰组和VP16组显著升高,差异有统计学意义(P<0.01),阴性对照组与空白对照组比较差异无统计学意义(P=0.848)。结论：联合WISP-2 siRNA和足叶乙甙(VP16)通过抑制U251细胞增殖、促进凋亡,增加U251细胞对VP16敏感性。更多还原

【Abstract】 Objective:To investigate the expression and significance of Wnt-1 induced secreted protein 2 (WISP-2) gene in human brain astrocytomas and its relationship with the genesis and development of human brain astrocytomas.Methods:The expression of WISP-2 mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) in 47 samples of human brain astrocytomas tissues, as well as 10 normal brain tissues.The correlation between WISP-2 expression levels of astrocytomas and its clinical indicators was analysed.Results:RT-PCR results showed that normal brain tissues had no WISP-2 mRNA expression.The relative expression level in astrocytomas was 0.677±0.445. Compared with the normal group there were statistically significant differences (t= 4.783, P<0.05). The relative expression levels of WISP-2 mRNA in high grade group (gradeⅢ-Ⅳ) and low grade group (gradeⅠ-Ⅱ) were 0.924±0.438,0.478±0.344, respectively.The difference was significant (t=3.909, P<0.05).The increase of WISP-2 correlated positively with tumor grade (r=0.448, P=0.002). The WISP-2 mRNA expression had no significant relationship with age, sex, tumor location (all P>0.05), but had a significant relationship with tumor size (P<0.05).Conclusion:WISP-2 mRNA could expresse in human brain astrocytomas and the expression was correlated with the malignant degree of human astrocytomas. WISP-2 mRNA may play an important regulatory role in the occurrence and development of human astrocytomas, which contributes to human brain astrocytomas malignant progression. Objective To investigate the expression and significance of WISP-2 protein in human brain astrocytomas and and its clinicopathologic significance.Methods The expression of WISP-2 protein of 54 human astrocytomas and 10 human normal brain tissues that were as control group was respectively examined. The expression of WISP-2 protein in these samples was detected using immunohistochemical staining. Statistical analysis was performed to investigate the ralation between WISP-2 protein expression and the clinicopathologic features.Results Immunohistochemical staining showed that the protein expression was undetected in normal brain tissues (0/10),but the positive rate of WISP-2 protein expression was 64.8%(35/54) in astrocytomas. There was significant difference between the positive rate of WISP-2 expression in astrocytomas and that of normal tissues(P<0.05). The positive rates of WISP-2 protein in high-grade group(gradeⅢ-Ⅳ) and low-grade group (gradeⅠ-Ⅱ) were 80.8%(21/26) and 50.0%(14/28), respectively.The significant difference was seen between high-grade (gradeⅢ-Ⅳ) and low-grade (gradeⅠ-Ⅱ) (P<0.05). With the combination of histopathological data, we found that the expression level of WISP-2 protein was closely correlated the differentiation degrees and tumor diameters of astrocytomas, but not with patients’ age, gender, and tumor location(all P>0.05).Conclusion The expression of WISP-2 is closely related to the malignant degree of human brain astrocytoms., and WISP-2 may be one of the malignant biomarkers in the pathogenesis and progression of human brain astrocytoms. Objective:To construct siRNA silencing human WISP-2 gene expression and filter out effective siRNA sequence.Methods:The expression of WISP-2 in the U251 was checked by RT-PCR first. The sequences targeting WISP-2 gene were designed and synthesized, then cloned into siRNA expression vectors. There combinant plasmids were transiently transfected into U251 cells via LipofectamineTM2000. The RT-PCR was used to examine mRNA expression of WISP-2 gene.Western Blot was used to detect expression of WISP-2 protein.And inverted fluorescence microscope was used to observe the effect of transfection.Results:WISP-2 mRNA expressed in U251 cell lines. RT-PCR and Western blot used to detect that WISP-2-siRNA-289 sequence inhibiting WISP-2 expression and had the best silencing effect. Observed by Inverted fluorescence microscope the transfection efficiency reached (87.03±11.56)%.Conclusion:The siRNA targeting WISP-2 gene can inhibit the expression ofKEYWORDS:siRNA, WISP-2, transfectionObjective:The present study was carried out to investigate the effect on the bionomics of U251 by the silence of WISP-2. Then we could figure out the function of WISP-2 during the proliferation and apoptosis of human astrocytomas.Methods:There were three groups in the experiment:blank control group(used the normal U251), negative control group(the inclusion of non-specific siRNA/ Lipofectamine complexes in U251 cell lines) and siRNA interference group (WISP-2-siRNA-289-liposome-transfected U251 cell line, which was contracted in the Part 3). Human astrocytic glioma cell line U251 was cultured in vitro, then MTT method, Transwell assay, and flow cytometer (FCM) analysis and Western Blot were applied to measure cell growth,cell invasiveness,apoptosis,expression of WISP-2,Bcl-2,and Bax protein.Results:Detected at the same time by MTT, the siRNA interference group OD was significantly higher than that of blank control group and negative control group (P<0.05). The result of Transwell assay exhibited that cell number through the artificial polycarbonate basal membrane became decreased compared with the control group after 48 hours(P<0.05). By the flow cytometry’s test, we found that the mean apoptotic rate of siRNA interference group was (16.59±1.40)%. Compared with the apoptotic rate of blank control group (3.13±0.34)% and the apoptotic rate of negative control group (3.42±0.48)%, the difference was statistically significant (P<0.01). The expression of WISP-2 protein in siRNA interference group(18.67±1.40) % was significantly lower than blank control group (70.18±1.82)% and negative control group (49.51±1.71)%(P<0.01). The expression of Bcl-2 protein in siRNA interference group(29.67±1.47)% was significantly lower than blank control group (49.51±1.71)% and negative control group (49.07±1.35)%(P<0.01). The expression of Bax protein in siRNA interference group(31.62±1.32)%was significantly higher than blank control group (23.98±1.47)% and negative control group (24.06±1.55)% (P<0.01).Correlation analysis showed that the expression of WISP-2 had a positive correlation with the expression of Bcl-2 protein. after siRNA silencing (r=0.995, P<0.05), and it had a negative correlation with the expression of Bax protein. after siRNA silencing (r=-0.944, P<0.05).Conclusion:The siRNA silencing expression of WISP-2 gene inhibits the proliferation and migration of U251 cells. and it increases the apoptosis of U251 cells. And it may by inhibiting expression of WISP-2 protein downregulate the ratio of Bcl-2/Bax to induce cell apoptosis. Objective:To evaluate the effect of combined therapy of Etoposide and WISP-2 siRNA on growth inhibition of human glioma U251 cells.Methods:U251 cells were transfected with WISP-2 siRNA via LipofectaminTM2000,and at the same time etoposide (VP16) was added into U251 cells medium. The proliferation and apoptosis of U251 cells were evaluated after 48 hours by MTT assay and flow cytometry.Results:Inhibitory effects of VP16 on proliferation of U251 cells were observed by MTT colorimetric survival assay. was which were in a time-and dose-dependent manner and the IC50 was 13.43μg/mL for 48 hours.Combined therapy of WISP-2 siRNA with VP16 inhibited proliferation of U251 cells.MTT assay showed that the the OD value of blank control group, negative control group, siRNA interference group, VP16 group and siRNA interference+VP16 group was 0.257±0.011、0.252±0.015、0.166±0.011、0.155±0.012、0.076±0.014, respectively. We find that the OD value of siRNA interference group and VP16 group was significantly lower comparing with that of blank control group and negative control group (P<0.05).The OD value of siRNA interference+VP16 group was significantly lower compared with that of siRNA interference group and the VP16 group (P<0.05), There were no significant difference between the blank control group and the negative control group (P>0.05).Combining therapy of WISP-2 siRNA and etoposide (VP16) significantly reduced the U251 cell proliferation. By the flow cytometry’s test, we found that the apoptotic rate of blank control group, negative control group,siRNA interference group, VP16 group and siRNA interference+VP16 group was (3.06±0.39)%, (3.28±0.57)%, (15.72±1.55)%, (14.76±1.41)%, (33.09±2.13)%, respectively. Statistical analysis showed the apoptotic rate of siRNA interference group and VP16 group was statistically significant higher than that of blank control group and negative control group.(P<0.01), and the apoptotic rate of siRNA interference+ VP16 group was statistically significant higher than that of siRNA interference group and VP16 group (P<0.05).There were no significant difference between the negative control group and the blank control group (P=0.848).Conclusion:The combination therapy of WISP-2 siRNA and etoposide could effectively improve thesensitivity of U251 cells to etoposide by inhibiting U251 cells’ proliferation and promoting U251’ apoptosis.更多还原