色素上皮衍生因子对黑素细胞生物学活性的影响
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摘要
第一部分良、恶性黑素细胞及组织中色素上皮衍生因子表达的研究
目的
探讨色素上皮衍生因子(PEDF)在皮肤良、恶性黑素细胞及组织中的表达及其意义。
方法
用免疫组化法检测正常皮肤组织、色素痣以及恶性黑素瘤组织中PEDF的表达与分布。用蛋白印迹法检测正常黑素细胞及恶性黑素瘤细胞株A375中PEDF蛋白的表达与分布。用RT-PCR法检测正常黑素细胞及恶性黑素瘤细胞株A375中PEDF mRNA的表达。
结果
1、PEDF蛋白在正常皮肤组织、色素痣以及恶性黑素瘤组织中的表达依次递减,三者差异在统计学上有非常显著意义(P＜0.001)。
2、PEDF蛋白在正常黑素细胞中有表达,恶性黑素瘤细胞株表达减弱在统计学上有非常显著意义(P＜0.001)。
3、PEDF mRNA在恶性黑素瘤细胞中的表达非常显著低于正常黑素细胞(P＜0.001)。
结论
恶性黑素瘤细胞PEDF蛋白和mRNA表达均降低,可能与恶性黑素瘤的发病有关。
第二部分siRNA沉默人黑素细胞色素上皮衍生因子基因表达的研究
目的
应用小干扰RNA(siRNA)干扰人黑素细胞的色素上皮衍生因子(PEDF)基因的表达,了解PEDF基因对黑素细胞增殖的作用。
方法
将PEDF siRNA经LipofectamineTM2000脂质体转染人黑素细胞后,以实时荧光定量RT-PCR和蛋白印迹法检测细胞PEDF mRNA和蛋白的表达,MTT法检测干扰后细胞的增殖水平。
结果
转染后黑素细胞的PEDF mRNA与蛋白表达明显减弱(P＜0.001)。黑素细胞增殖率在转染后显著升高(P＜0.001)。
结论
PEDF小片段干扰RNA可显著抑制黑素细胞中PEDF基因的表达;PEDF对黑素细胞的过度增殖有抑制作用。
第三部分人恶性黑素瘤细胞株中色素上皮衍生因子基因的突变研究
目的
探讨色素上皮衍生因子(PEDF)基因在恶性黑素瘤细胞系中的突变状况。
方法
采用聚合酶链反应一单链构象多态性分析(PCR-SSCP)对人恶性黑素瘤细胞株A375中PEDF基因的所有八个外显子进行突变检测。对SSCP分析有异常泳动条带样本的PCR产物进行DNA测序。
结果
恶性黑色素瘤细胞株A375与正常黑素细胞相比,第3到第7外显子都出现了DNA泳动变位,其中以外显子5,6最为明显。PEDF基因第5,6外显子均存在突变:第5外显子的突变类型以单个碱基的缺失为主,第6外显子的突变类型则以单个碱基的突变为主。
结论
PEDF基因的突变可能在恶性黑素瘤的发病中起一定作用。
PartⅠExpression of Pigment Epithelium Derived Factor In Human Melanocytes and Malignant Melanoma Cells And Tissues
Objective
To study the production and distribution of pigment epithelium derived factor (PEDF) in human melanocytes and malignant melanoma cells and tissues.
Methods
The expression and distribution of PEDF in healthy skins,pigmented nevus and human malignant melanoma tissues were detected by immunohistochemical method.The expression of PEDF protein in human melanocytes and malignant melanoma A375 cells were detected by Western blot analysis.The expression of PEDF mRNA values in human melanocytes and malignant melanoma A375 cells were measured by reverse transcription polymerase chain reaction(RT-PCR).
Results
PEDF could be expressed by human melanocytes.The expression of PEDF protein got lower and lower in healthy skins,pigmented nevus and human malignant melanoma tissues(P＜0.001).The expression of PEDF protein was observed in melanocytes and was almost absent in malignant melanoma A375 cells(P＜0.001).The expression of PEDF mRNA was much lower in malignant melanoma A375 cells than human melanocytes(P＜0.001).
Conclusions
The lower expression of PEDF in malignant melanoma may contribute to its pathogenesis.
PartⅡEffects of Pigment Epithelium Derived Factor Inhibition by siRNA on human melanocytes
Objective
To evaluate the effect of small interferring RNA(siRNA) targeting pigment epithelium derived factor(PEDF) gene on the proliferation of human melanocytes.
Methods
After siRNA for PEDF gene was transfected into human melanocytes by LipofectimineTM2000,PEDF gene expression was assessed by real-time quantitive PCR,and PEDF protein expression was detected by Western blot analysis.MTT assay was used to assess the proliferation of the cells.
Results
The expression of PEDF gene in human melanocytes could be effectively suppressed by siRNA(P＜0.001) and the proliferation rate of human melanocytes was elevateed markedly(P＜0.001).
Conclusions
The expression of PEDF gene in human melanocytes can be significantly decreased by RNAi.PEDF can inhibit exorbitant proliferation of human melanocytes.
PartⅢDetection of Pigment Epithelium Derived Factor Gene Mutations In Human Malignant Melanoma Cell Line
Objective
To discuss the role of pigment epithelium derived factor(PEDF) gene in the pathogenesis of malignant melanoma.
Methods
All the eight exons of PEDF gene were scanned in human malignant melanoma cell line A375 by single strand conformation polymorphism analysis ofpolymerase chain reaction products(PCR-SSCP),followed by DNA sequencing.
Results
SSCP analysis of the genomic DNA from all the eight exons of PEDF gene demonstrated several polymorphisms in exons 3,4,5,6 and 7,especially in exon 5,6. Frame-shift mutation was detected in exon 5 and point mutation was detected in exon 6.
Conclusions
Mution of PEDF gene may contribute to the pathogenesis of human malignant melanoma.
目的
探讨色素上皮衍生因子(PEDF)在皮肤良、恶性黑素细胞及组织中的表达及其意义。
方法
用免疫组化法检测正常皮肤组织、色素痣以及恶性黑素瘤组织中PEDF的表达与分布。用蛋白印迹法检测正常黑素细胞及恶性黑素瘤细胞株A375中PEDF蛋白的表达与分布。用RT-PCR法检测正常黑素细胞及恶性黑素瘤细胞株A375中PEDF mRNA的表达。
结果
1、PEDF蛋白在正常皮肤组织、色素痣以及恶性黑素瘤组织中的表达依次递减,三者差异在统计学上有非常显著意义(P＜0.001)。
2、PEDF蛋白在正常黑素细胞中有表达,恶性黑素瘤细胞株表达减弱在统计学上有非常显著意义(P＜0.001)。
3、PEDF mRNA在恶性黑素瘤细胞中的表达非常显著低于正常黑素细胞(P＜0.001)。
结论
恶性黑素瘤细胞PEDF蛋白和mRNA表达均降低,可能与恶性黑素瘤的发病有关。
第二部分siRNA沉默人黑素细胞色素上皮衍生因子基因表达的研究
目的
应用小干扰RNA(siRNA)干扰人黑素细胞的色素上皮衍生因子(PEDF)基因的表达,了解PEDF基因对黑素细胞增殖的作用。
方法
将PEDF siRNA经LipofectamineTM2000脂质体转染人黑素细胞后,以实时荧光定量RT-PCR和蛋白印迹法检测细胞PEDF mRNA和蛋白的表达,MTT法检测干扰后细胞的增殖水平。
结果
转染后黑素细胞的PEDF mRNA与蛋白表达明显减弱(P＜0.001)。黑素细胞增殖率在转染后显著升高(P＜0.001)。
结论
PEDF小片段干扰RNA可显著抑制黑素细胞中PEDF基因的表达;PEDF对黑素细胞的过度增殖有抑制作用。
第三部分人恶性黑素瘤细胞株中色素上皮衍生因子基因的突变研究
目的
探讨色素上皮衍生因子(PEDF)基因在恶性黑素瘤细胞系中的突变状况。
方法
采用聚合酶链反应一单链构象多态性分析(PCR-SSCP)对人恶性黑素瘤细胞株A375中PEDF基因的所有八个外显子进行突变检测。对SSCP分析有异常泳动条带样本的PCR产物进行DNA测序。
结果
恶性黑色素瘤细胞株A375与正常黑素细胞相比,第3到第7外显子都出现了DNA泳动变位,其中以外显子5,6最为明显。PEDF基因第5,6外显子均存在突变:第5外显子的突变类型以单个碱基的缺失为主,第6外显子的突变类型则以单个碱基的突变为主。
结论
PEDF基因的突变可能在恶性黑素瘤的发病中起一定作用。
PartⅠExpression of Pigment Epithelium Derived Factor In Human Melanocytes and Malignant Melanoma Cells And Tissues
Objective
To study the production and distribution of pigment epithelium derived factor (PEDF) in human melanocytes and malignant melanoma cells and tissues.
Methods
The expression and distribution of PEDF in healthy skins,pigmented nevus and human malignant melanoma tissues were detected by immunohistochemical method.The expression of PEDF protein in human melanocytes and malignant melanoma A375 cells were detected by Western blot analysis.The expression of PEDF mRNA values in human melanocytes and malignant melanoma A375 cells were measured by reverse transcription polymerase chain reaction(RT-PCR).
Results
PEDF could be expressed by human melanocytes.The expression of PEDF protein got lower and lower in healthy skins,pigmented nevus and human malignant melanoma tissues(P＜0.001).The expression of PEDF protein was observed in melanocytes and was almost absent in malignant melanoma A375 cells(P＜0.001).The expression of PEDF mRNA was much lower in malignant melanoma A375 cells than human melanocytes(P＜0.001).
Conclusions
The lower expression of PEDF in malignant melanoma may contribute to its pathogenesis.
PartⅡEffects of Pigment Epithelium Derived Factor Inhibition by siRNA on human melanocytes
Objective
To evaluate the effect of small interferring RNA(siRNA) targeting pigment epithelium derived factor(PEDF) gene on the proliferation of human melanocytes.
Methods
After siRNA for PEDF gene was transfected into human melanocytes by LipofectimineTM2000,PEDF gene expression was assessed by real-time quantitive PCR,and PEDF protein expression was detected by Western blot analysis.MTT assay was used to assess the proliferation of the cells.
Results
The expression of PEDF gene in human melanocytes could be effectively suppressed by siRNA(P＜0.001) and the proliferation rate of human melanocytes was elevateed markedly(P＜0.001).
Conclusions
The expression of PEDF gene in human melanocytes can be significantly decreased by RNAi.PEDF can inhibit exorbitant proliferation of human melanocytes.
PartⅢDetection of Pigment Epithelium Derived Factor Gene Mutations In Human Malignant Melanoma Cell Line
Objective
To discuss the role of pigment epithelium derived factor(PEDF) gene in the pathogenesis of malignant melanoma.
Methods
All the eight exons of PEDF gene were scanned in human malignant melanoma cell line A375 by single strand conformation polymorphism analysis ofpolymerase chain reaction products(PCR-SSCP),followed by DNA sequencing.
Results
SSCP analysis of the genomic DNA from all the eight exons of PEDF gene demonstrated several polymorphisms in exons 3,4,5,6 and 7,especially in exon 5,6. Frame-shift mutation was detected in exon 5 and point mutation was detected in exon 6.
Conclusions
Mution of PEDF gene may contribute to the pathogenesis of human malignant melanoma.
引文
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