BAI-1在膀胱癌中的表达及对肿瘤血管抑制作用的研究
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摘要
第一部分BAI-1在人膀胱移行上皮细胞癌组织中的表达及与P53、MVD、VEGF的相关性研究
目的探讨BAI-1在不同分期膀胱移行上皮细胞癌(bladder transitional cell carcinoma、BTCC)中的表达差异以及BAI-1抑制肿瘤血管内皮增殖的机制。
方法取人BTCC石蜡切片标本131例,正常膀胱粘膜组织石蜡切片标本28例。正常膀胱粘膜组织切片标本为对照组,人BTCC组织切片标本为研究组。采用免疫组织化学SP法分别检测BAI-1、VEGF、MVD、P53,进行半定量统计学分析。取临床新鲜BTCC组织标本21例,正常膀胱粘膜组织9例。应用Western blot方法分析BAI-1在BTCC组织及正常膀胱粘膜组织中的表达差异。
结果免疫组化统计分析及western blot条带显示正常膀胱粘膜组织中BAI-1表达明显高于BTCC组织,并且与临床分期相关,T1局限期BTCC组织中BAI-1表达远高于T2-4进展期BTCC组织(P<0.05)。同时可以看到BAI-1表达与VEGF且有反相关性(r=-0.661,P=0.000)；BAI-1与MVD表达具有反相关性(r=-0.406,P=0.002)；BAI-1与P53表达具有反相关性(r=-0.675,P=0.000)。
结论在BTCC组织中BAI-1含量较正常膀胱粘膜组织明显降低且与BTCC临床分期相关；BAI-1表达与VEGF、MVD成负相关,与突变型P53成负相关性。表明BAI-1可能参与了肿瘤微血管增殖的负调节,而其BAI-1的表达减少也可能与P53的突变相关。
第二部分重组质粒载体pReceiver-M61-BAI1转染T24与HUVEC细胞株的效应研究
目的构建并鉴定含有BAI-1基因的脂质体质粒载体pReceiver-M61-BAI1,并感染人膀胱癌细胞T24及人脐静脉血管内皮细胞HUVEC,研究其对T24和HUVEC生物学影响。
方法将携有BAI-1基因的重组真核表达质粒pReceiver-M61-BAI1转化大肠杆菌DH5a并扩增,抽提纯化质粒并进行酶切鉴定,pReceiver-M61-BAI1体外转染T24和HUVEC细胞,筛选稳定转染的细胞并扩增培养,以转染了空质粒的T24和HUVEC细胞为对照,用Q-qPCR及Western blot检测基因mRNA及蛋白表达水平。应用流式细胞仪分别检测BAI1基因转染前后T24细胞和HUVEC细胞凋亡情况的变化,MTT法检测BAI-1基因转染前后T24细胞和HUVEC细胞的增殖情况。
结果pReceiver-M61-BAI1的酶切鉴定证实含有目的基因BAI-1；T24和HUVEC细胞株在转染pReceiver-M61-BAI1为研究组,转染pReceiver-M61为对照组,利用Q-PCR技术检测,发现Q-PCR产物经凝胶电泳有明显的阳性条带,而对照组则无明显阳性条带；Western blot检测证实转染pReceiver-M61-BAI1的T24细胞和HUVEC细胞的BAI-1蛋白表达呈阳性；MTT检测发现HUVEC细胞在转染pReceiver-M61-BAI1后12、48、72h的吸光度值明显低于转染pReceiver-M61的对照组以及转染了pReceiver-M61-BAI1的T24细胞组。流式细胞学检测证实转染BAI-1的HUVEC凋亡率明显高于对照组和转染pReceiver-M61-BAI1的T24组。
结论BAI-1具有明显的血管内皮细胞抑制作用,而在体外实验中未发现对膀胱癌T24细胞具有直接抑制作用。
Part I The expression of BAI-1in the bladder transitional cell carcinoma tissues and the correlation between BAI-1and p53、MVDand VEGF
Objective:To investigate the different expression of different stages of BTCC. To investigate the mechanism of BAI-1the inhibits the proliferation of tumor endothelial cell.
Methods:Paraffin section preparation of131cases of BTCC and28cases of normal bladder mucosal tissue were taken. Normal bladder mucosal tissue section was set as control group and BTCC tissue section was set as observe group. Immunohistochemical SP method was used to detect the expression of BAI-1, VEGF, MVD and p53, and semi-quantitative statistically analysis was use.21cases of BTCC and28cases of normal bladder mucosal tissue of fresh tissue samples were taken and western blot was used to detect the different expression of BAI-1between them.
Result:The result of Immunohistochemical staining and western blot showed that the expression of BAI-1in normal bladder mucosal tissue was higher than that in BTCC tissue and the expression of BAI-1was correlated to clinical stages. The expression of BAI-1in BTCC tissue of T1stage was much higher than that of T2-4(P <0.05). The expression of BAI-1had negative relationship to the expression of VEGF (r=-0.661, P=0.000). The expression of BAI-1had negative relationship to the expression of MVD (r=-0.406, P=0.002). The expression of BAI-1had negative relationship to the expression of mutant type P53(r=-0.675,P=0.000)
Conclusion:The content of BAI-1in BTCC tissue is much lower than that in normal bladder mucosal tissue. The content of BAI-1is related to the clinical stage of BTCC. The expression of BAI-1had negative relationship to the expression of VEGF, MVD and mutant type P53, which shows that BAI-1may participate in the negative regulation of proliferation of tumor micrangium. The reduction of the expression of BAI-1may relate to the mutant of P53.
Part II the study of recombinant plasmid vector pReceiver-M61-BAI1transfect T24and HUVEC
Objective:To construct and identify recombinant plasmid vector pReceiver-M61-BAI1that includes the gene of BAI-1for studying its role in human carcinoma cells of urinary bladder (T24) and human vascular endothelial cell of umbilical vein (HUVEC).
Methods:Recombinant eukaryotic expression vector pReceiver-M61-BAI1was transformed and indentified. pReceiver-M61-BAI1was used to transfect T24and HUVEC and stable transfected cells were cultured. Null vector transfection was used as the control. RT-qPCR and Western blot were used to detect the expression of gene and protein. Flow cytometry was used to study the apoptosis of the T24and HUVEC before and after gene transfection. MTT was used to study the proliferation of the T24and HUVEC before and after gene transfection.
Result:pReceiver-M61-BAI1identified by restriction enzyme cleave was verified including BAI-1gene. The RT-PCR product of pReceiver-M61-BAI1-T24cell and pReceiver-M61-BAI1-HUVEC cell showed obvious positive stripe by gel electrophoresis,which was not observed in the control group. The result of Western blot showed that the positive cells rate of BAI-1in pReceiver-M61-BAI1-T24cell and pReceiver-M61-BAI1-HUVEC cell were much higher than that of the control group. The result of MTT showed that the absorbance value A of the pReceiver-M61-BAI1-HUVEC cell at the second day, the third day and forth day were much lower than that of the control group and pReceiver-M61-BAI1-T24cell group. The result of flow cytometry showed that the apoptosis rate of HUVEC transfected BAI-1was much higher than that of the control group and the pReceiver-M61-BAI1-T24cell group.
Conclusion:BAI-1has obviously inhibited effect on the vascular endothelial cells. While directed inhibited effect of BAI-1on T24cells is not found in vitro.
目的探讨BAI-1在不同分期膀胱移行上皮细胞癌(bladder transitional cell carcinoma、BTCC)中的表达差异以及BAI-1抑制肿瘤血管内皮增殖的机制。
方法取人BTCC石蜡切片标本131例,正常膀胱粘膜组织石蜡切片标本28例。正常膀胱粘膜组织切片标本为对照组,人BTCC组织切片标本为研究组。采用免疫组织化学SP法分别检测BAI-1、VEGF、MVD、P53,进行半定量统计学分析。取临床新鲜BTCC组织标本21例,正常膀胱粘膜组织9例。应用Western blot方法分析BAI-1在BTCC组织及正常膀胱粘膜组织中的表达差异。
结果免疫组化统计分析及western blot条带显示正常膀胱粘膜组织中BAI-1表达明显高于BTCC组织,并且与临床分期相关,T1局限期BTCC组织中BAI-1表达远高于T2-4进展期BTCC组织(P<0.05)。同时可以看到BAI-1表达与VEGF且有反相关性(r=-0.661,P=0.000)；BAI-1与MVD表达具有反相关性(r=-0.406,P=0.002)；BAI-1与P53表达具有反相关性(r=-0.675,P=0.000)。
结论在BTCC组织中BAI-1含量较正常膀胱粘膜组织明显降低且与BTCC临床分期相关；BAI-1表达与VEGF、MVD成负相关,与突变型P53成负相关性。表明BAI-1可能参与了肿瘤微血管增殖的负调节,而其BAI-1的表达减少也可能与P53的突变相关。
第二部分重组质粒载体pReceiver-M61-BAI1转染T24与HUVEC细胞株的效应研究
目的构建并鉴定含有BAI-1基因的脂质体质粒载体pReceiver-M61-BAI1,并感染人膀胱癌细胞T24及人脐静脉血管内皮细胞HUVEC,研究其对T24和HUVEC生物学影响。
方法将携有BAI-1基因的重组真核表达质粒pReceiver-M61-BAI1转化大肠杆菌DH5a并扩增,抽提纯化质粒并进行酶切鉴定,pReceiver-M61-BAI1体外转染T24和HUVEC细胞,筛选稳定转染的细胞并扩增培养,以转染了空质粒的T24和HUVEC细胞为对照,用Q-qPCR及Western blot检测基因mRNA及蛋白表达水平。应用流式细胞仪分别检测BAI1基因转染前后T24细胞和HUVEC细胞凋亡情况的变化,MTT法检测BAI-1基因转染前后T24细胞和HUVEC细胞的增殖情况。
结果pReceiver-M61-BAI1的酶切鉴定证实含有目的基因BAI-1；T24和HUVEC细胞株在转染pReceiver-M61-BAI1为研究组,转染pReceiver-M61为对照组,利用Q-PCR技术检测,发现Q-PCR产物经凝胶电泳有明显的阳性条带,而对照组则无明显阳性条带；Western blot检测证实转染pReceiver-M61-BAI1的T24细胞和HUVEC细胞的BAI-1蛋白表达呈阳性；MTT检测发现HUVEC细胞在转染pReceiver-M61-BAI1后12、48、72h的吸光度值明显低于转染pReceiver-M61的对照组以及转染了pReceiver-M61-BAI1的T24细胞组。流式细胞学检测证实转染BAI-1的HUVEC凋亡率明显高于对照组和转染pReceiver-M61-BAI1的T24组。
结论BAI-1具有明显的血管内皮细胞抑制作用,而在体外实验中未发现对膀胱癌T24细胞具有直接抑制作用。
Part I The expression of BAI-1in the bladder transitional cell carcinoma tissues and the correlation between BAI-1and p53、MVDand VEGF
Objective:To investigate the different expression of different stages of BTCC. To investigate the mechanism of BAI-1the inhibits the proliferation of tumor endothelial cell.
Methods:Paraffin section preparation of131cases of BTCC and28cases of normal bladder mucosal tissue were taken. Normal bladder mucosal tissue section was set as control group and BTCC tissue section was set as observe group. Immunohistochemical SP method was used to detect the expression of BAI-1, VEGF, MVD and p53, and semi-quantitative statistically analysis was use.21cases of BTCC and28cases of normal bladder mucosal tissue of fresh tissue samples were taken and western blot was used to detect the different expression of BAI-1between them.
Result:The result of Immunohistochemical staining and western blot showed that the expression of BAI-1in normal bladder mucosal tissue was higher than that in BTCC tissue and the expression of BAI-1was correlated to clinical stages. The expression of BAI-1in BTCC tissue of T1stage was much higher than that of T2-4(P <0.05). The expression of BAI-1had negative relationship to the expression of VEGF (r=-0.661, P=0.000). The expression of BAI-1had negative relationship to the expression of MVD (r=-0.406, P=0.002). The expression of BAI-1had negative relationship to the expression of mutant type P53(r=-0.675,P=0.000)
Conclusion:The content of BAI-1in BTCC tissue is much lower than that in normal bladder mucosal tissue. The content of BAI-1is related to the clinical stage of BTCC. The expression of BAI-1had negative relationship to the expression of VEGF, MVD and mutant type P53, which shows that BAI-1may participate in the negative regulation of proliferation of tumor micrangium. The reduction of the expression of BAI-1may relate to the mutant of P53.
Part II the study of recombinant plasmid vector pReceiver-M61-BAI1transfect T24and HUVEC
Objective:To construct and identify recombinant plasmid vector pReceiver-M61-BAI1that includes the gene of BAI-1for studying its role in human carcinoma cells of urinary bladder (T24) and human vascular endothelial cell of umbilical vein (HUVEC).
Methods:Recombinant eukaryotic expression vector pReceiver-M61-BAI1was transformed and indentified. pReceiver-M61-BAI1was used to transfect T24and HUVEC and stable transfected cells were cultured. Null vector transfection was used as the control. RT-qPCR and Western blot were used to detect the expression of gene and protein. Flow cytometry was used to study the apoptosis of the T24and HUVEC before and after gene transfection. MTT was used to study the proliferation of the T24and HUVEC before and after gene transfection.
Result:pReceiver-M61-BAI1identified by restriction enzyme cleave was verified including BAI-1gene. The RT-PCR product of pReceiver-M61-BAI1-T24cell and pReceiver-M61-BAI1-HUVEC cell showed obvious positive stripe by gel electrophoresis,which was not observed in the control group. The result of Western blot showed that the positive cells rate of BAI-1in pReceiver-M61-BAI1-T24cell and pReceiver-M61-BAI1-HUVEC cell were much higher than that of the control group. The result of MTT showed that the absorbance value A of the pReceiver-M61-BAI1-HUVEC cell at the second day, the third day and forth day were much lower than that of the control group and pReceiver-M61-BAI1-T24cell group. The result of flow cytometry showed that the apoptosis rate of HUVEC transfected BAI-1was much higher than that of the control group and the pReceiver-M61-BAI1-T24cell group.
Conclusion:BAI-1has obviously inhibited effect on the vascular endothelial cells. While directed inhibited effect of BAI-1on T24cells is not found in vitro.
引文
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