人Canstatin基因转染对肝癌HepG2细胞血管生成拟态的影响及机制研究
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摘要
目的:通过肝癌HepG2细胞的体外三维培养,观察其形成血管生成拟态的能力,探讨重组人Canstatin基因表达产物对肝癌HepG2细胞血管生成拟态形成能力的影响及作用机制。
方法：将pcDNA3.1-Canstatin-3Flag分泌型重组质粒,用Cacl2法转染到大肠杆菌DH5a,经AMP筛选,挑取阳性质粒酶切鉴定、测序,用脂质载体介导法将pcDNA3.1-Canstatin"3Flag转染人肝癌HepG2细胞,以空载体pcDNA3.1转染肝癌HepG2细胞为对照,经G418培养基筛选获得阳性克隆,Western-blot鉴定转染细胞中Canstatin蛋白的表达。建立HepG2细胞基底膜基质凝胶体外三维细胞培养模型,以HepG2细胞在六孔板中常规培养为对照,观察HepG2细胞血管生成拟态形成能力。各组细胞(HepG2细胞组、空载体转染HepG2细胞组、重组载体转染HepG2细胞组)分别在96孔板中常规培养48h,MTT法检测各组增殖抑制率。各组细胞在基底膜基质凝胶进行体外三维培养,观察比较各组细胞的血管生成拟态形成能力,同时用免疫细胞化学法检测各组细胞MMp-2、VE-cadherin蛋白表达情况。
结果：成功获得684bp人Canstatin基因,测序证明与Genbank中比对一致。PCR可检测出重组载体转染人HepG2细胞基因组中存在Canstatin基因,Western-blot可检测出Canstatin基因在重组载体转染人HepG2细胞中有特异性表达。人肝癌HepG2细胞在体外三维培养72h,直接在倒置显微镜下观察,可见细胞呈长梭形,周边长出多个伪足,彼此之间形成网状连接,形成环状或管状结构(血管生成拟态VM),而人肝癌HepG2细胞在六孔板中常规培养下贴壁生长,未见VM结构。MTT法检测HepG2细胞组、空载体转染HepG2细胞组、重组载体转染HepG2细胞组490nm波长吸光度值并计算增殖抑制率分别为：0%、3.57%、14.80%。倒置显微镜下(x200)随机取上、下、左、右、中5个视野记录环状结构的数量,重组载体转染组VM结构的计数为(2.67±2.08)个,低于HepG2细胞组(9.67±3.51)个(P<0.01),与空质粒转染组(11.00±3.61)个对比有统计学意义(P<0.01),空质粒转染组与HepG2细胞组对比无统计学意义(P>0.01)。免疫细胞化学法检测各组细胞VE-cadherin蛋白表达灰度值为：重组载体转染组细胞(5.63±0.42),低于空载体转染组(14.58±0.72)(P<0.01),同时也低于HepG2细胞组(16.73±1.32)(P<0.01),空载体转染组和HepG2细胞组VE-cadherin蛋白表达灰度值无差异(P>0.01)。HepG2组、空载体转染HepG2组、重组载体转染HepG2组MMp-2蛋白表达灰度值分别为27.53±1.03、25.72±0.83、27.02±2.13,各组间P值无显著性差异(P>0.01)。
结论：1.人肝癌HepG2细胞株在体外常规培养下未见VM结构,在体外三维培养条件下具有血管生成拟态的形成能力；2.成功将pcDNA3.1-Canstatin-3Flag分泌型重组质粒转染至人HepG2细胞中,细胞中有Canstatin蛋白表达；3Canstatin蛋白可抑制人肝癌HepG2细胞的增殖；4Canstatin蛋白可抑制人肝癌HepG2细胞血管生成拟态的形成能力；5Canstatin蛋白可抑制体外三维培养条件下HepG2细胞VE-cadherin蛋白的表达,对MMp2的表达没有影响。
Object:Hepatoma cell line HepG2 in vitro culture, to observe the formation of vascular mimicry, and to observe and explore the gene expression product of recombinant human Canstatin hepatoma HepG2 cells on vasculogenic mimicry in the formation of a very possible mechanism.
Methods:pcDNA3.1-Canstatin-3Flag secreted recombinant plasmid transfection with Cacl2 into E.coli DH5a, selected by AMP, picked plasmid restriction endonuclease digestion, sequencing, lipid vector-mediated method with pcDNA3.1- Canstatin-3Flag transfected into human hepatoma HepG2 cells, and use the empty vector pcDNA3.1 transfected hepatoma HepG2 cells as control, G418 medium screened positive clones, Western-blot identification of transfected cells Canstatin protein. The establishment of artificial basement membrane matrix gel HepG2 cells in vitro three-dimensional cell culture model, HepG2 cells were cultured in six-well plate as a control on the formation of vasculogenic mimicry in HepG2 ability to detect. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were routinely cultured in 96 well plate 48h, MTT assay inhibition rate of value added in each group. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were in the artificial basement membrane matrix gel in vitro culture, and compared three groups of cells forming ability of the pipeline, the formation of immunocytochemical detection of vascular mimicry The three groups of cells MMp-2, VE-cadherin protein expression in the case.
Results:The successful person canstatin 684bp gene sequencing that in comparison with the same Genbank. PCR can detect the recombinant vector was transfected into human HepG2 cells genome contains genes Canstatin, Western-blot to detect Canstatin gene in recombinant plasmid transfected into HepG2 cells with specific expression. Human hepatoma HepG2 cells cultured under in vitro culture 72h, elongated spindle cells can be observed, many pseudopodia grow around each other and form a network connection, a ring or tube in human hepatocellular carcinoma HepG2 cells were cultured in six-well plate under adherent growth, can not form a ring or tube. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were routinely cultured in 96 well plate 48h, MTT assay inhibition rate of value added in each group were:0%,3.57%,14.80%. To HepG2 group, empty vector transfected HepG2 group, recombinant vector transfected HepG2 group while in vitro culture,72h after the three cells can form tubular structures, select the VM under a microscope relatively concentrated area,200 times the microscope counting, re- vector transfection group structure The number of VM (2.67±2.08) were lower than the parental HepG2 cell group (9.67±3.51) months (P<0.01), empty vector transfected group The number of VM structures (11.00±3.61) a, and the parental HepG2 cell group (9.67±3.51) showed no significant meaning (P> 0.01). Recombinant vector transfected cells expression of VE-cadherin gray value (5.63±0.42) lower than the empty vector group (14.58±0.72) and HepG2 cell group (16.73±1.32) (P<0.01). HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 group MMp-2 protein expression in gray values were 27.53±1.03,25.72±0.83,27.02±2.13 (P> 0.01).
Conclusion:1. Human hepatoma HepG2 cells were cultured in vitro can not be formed under the structure of vasculogenic mimicry in vitro culture conditions can form vasculogenic mimicry
2.Successfully pcDNA3.1-Canstatin-3Flag secreted recombinant plasmid was transfected into HepG2 cells, its typical and can be expressed protein Canstatin
3.Canstatin protein inhibits human hepatoma HepG2 cells value
4.Canstatin protein inhibits in vitro culture conditions in human hepatoma HepG2 cells the formation of vasculogenic mimicry
5.Canstatin protein inhibit in vitro culture conditions, HepG2 cells the expression of VE-cadherin protein, but no effect on the expression of MMp2.
方法：将pcDNA3.1-Canstatin-3Flag分泌型重组质粒,用Cacl2法转染到大肠杆菌DH5a,经AMP筛选,挑取阳性质粒酶切鉴定、测序,用脂质载体介导法将pcDNA3.1-Canstatin"3Flag转染人肝癌HepG2细胞,以空载体pcDNA3.1转染肝癌HepG2细胞为对照,经G418培养基筛选获得阳性克隆,Western-blot鉴定转染细胞中Canstatin蛋白的表达。建立HepG2细胞基底膜基质凝胶体外三维细胞培养模型,以HepG2细胞在六孔板中常规培养为对照,观察HepG2细胞血管生成拟态形成能力。各组细胞(HepG2细胞组、空载体转染HepG2细胞组、重组载体转染HepG2细胞组)分别在96孔板中常规培养48h,MTT法检测各组增殖抑制率。各组细胞在基底膜基质凝胶进行体外三维培养,观察比较各组细胞的血管生成拟态形成能力,同时用免疫细胞化学法检测各组细胞MMp-2、VE-cadherin蛋白表达情况。
结果：成功获得684bp人Canstatin基因,测序证明与Genbank中比对一致。PCR可检测出重组载体转染人HepG2细胞基因组中存在Canstatin基因,Western-blot可检测出Canstatin基因在重组载体转染人HepG2细胞中有特异性表达。人肝癌HepG2细胞在体外三维培养72h,直接在倒置显微镜下观察,可见细胞呈长梭形,周边长出多个伪足,彼此之间形成网状连接,形成环状或管状结构(血管生成拟态VM),而人肝癌HepG2细胞在六孔板中常规培养下贴壁生长,未见VM结构。MTT法检测HepG2细胞组、空载体转染HepG2细胞组、重组载体转染HepG2细胞组490nm波长吸光度值并计算增殖抑制率分别为：0%、3.57%、14.80%。倒置显微镜下(x200)随机取上、下、左、右、中5个视野记录环状结构的数量,重组载体转染组VM结构的计数为(2.67±2.08)个,低于HepG2细胞组(9.67±3.51)个(P<0.01),与空质粒转染组(11.00±3.61)个对比有统计学意义(P<0.01),空质粒转染组与HepG2细胞组对比无统计学意义(P>0.01)。免疫细胞化学法检测各组细胞VE-cadherin蛋白表达灰度值为：重组载体转染组细胞(5.63±0.42),低于空载体转染组(14.58±0.72)(P<0.01),同时也低于HepG2细胞组(16.73±1.32)(P<0.01),空载体转染组和HepG2细胞组VE-cadherin蛋白表达灰度值无差异(P>0.01)。HepG2组、空载体转染HepG2组、重组载体转染HepG2组MMp-2蛋白表达灰度值分别为27.53±1.03、25.72±0.83、27.02±2.13,各组间P值无显著性差异(P>0.01)。
结论：1.人肝癌HepG2细胞株在体外常规培养下未见VM结构,在体外三维培养条件下具有血管生成拟态的形成能力；2.成功将pcDNA3.1-Canstatin-3Flag分泌型重组质粒转染至人HepG2细胞中,细胞中有Canstatin蛋白表达；3Canstatin蛋白可抑制人肝癌HepG2细胞的增殖；4Canstatin蛋白可抑制人肝癌HepG2细胞血管生成拟态的形成能力；5Canstatin蛋白可抑制体外三维培养条件下HepG2细胞VE-cadherin蛋白的表达,对MMp2的表达没有影响。
Object:Hepatoma cell line HepG2 in vitro culture, to observe the formation of vascular mimicry, and to observe and explore the gene expression product of recombinant human Canstatin hepatoma HepG2 cells on vasculogenic mimicry in the formation of a very possible mechanism.
Methods:pcDNA3.1-Canstatin-3Flag secreted recombinant plasmid transfection with Cacl2 into E.coli DH5a, selected by AMP, picked plasmid restriction endonuclease digestion, sequencing, lipid vector-mediated method with pcDNA3.1- Canstatin-3Flag transfected into human hepatoma HepG2 cells, and use the empty vector pcDNA3.1 transfected hepatoma HepG2 cells as control, G418 medium screened positive clones, Western-blot identification of transfected cells Canstatin protein. The establishment of artificial basement membrane matrix gel HepG2 cells in vitro three-dimensional cell culture model, HepG2 cells were cultured in six-well plate as a control on the formation of vasculogenic mimicry in HepG2 ability to detect. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were routinely cultured in 96 well plate 48h, MTT assay inhibition rate of value added in each group. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were in the artificial basement membrane matrix gel in vitro culture, and compared three groups of cells forming ability of the pipeline, the formation of immunocytochemical detection of vascular mimicry The three groups of cells MMp-2, VE-cadherin protein expression in the case.
Results:The successful person canstatin 684bp gene sequencing that in comparison with the same Genbank. PCR can detect the recombinant vector was transfected into human HepG2 cells genome contains genes Canstatin, Western-blot to detect Canstatin gene in recombinant plasmid transfected into HepG2 cells with specific expression. Human hepatoma HepG2 cells cultured under in vitro culture 72h, elongated spindle cells can be observed, many pseudopodia grow around each other and form a network connection, a ring or tube in human hepatocellular carcinoma HepG2 cells were cultured in six-well plate under adherent growth, can not form a ring or tube. To HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 cells were routinely cultured in 96 well plate 48h, MTT assay inhibition rate of value added in each group were:0%,3.57%,14.80%. To HepG2 group, empty vector transfected HepG2 group, recombinant vector transfected HepG2 group while in vitro culture,72h after the three cells can form tubular structures, select the VM under a microscope relatively concentrated area,200 times the microscope counting, re- vector transfection group structure The number of VM (2.67±2.08) were lower than the parental HepG2 cell group (9.67±3.51) months (P<0.01), empty vector transfected group The number of VM structures (11.00±3.61) a, and the parental HepG2 cell group (9.67±3.51) showed no significant meaning (P> 0.01). Recombinant vector transfected cells expression of VE-cadherin gray value (5.63±0.42) lower than the empty vector group (14.58±0.72) and HepG2 cell group (16.73±1.32) (P<0.01). HepG2 group, empty vector transfected HepG2 group, recombinant vector was transfected into HepG2 group MMp-2 protein expression in gray values were 27.53±1.03,25.72±0.83,27.02±2.13 (P> 0.01).
Conclusion:1. Human hepatoma HepG2 cells were cultured in vitro can not be formed under the structure of vasculogenic mimicry in vitro culture conditions can form vasculogenic mimicry
2.Successfully pcDNA3.1-Canstatin-3Flag secreted recombinant plasmid was transfected into HepG2 cells, its typical and can be expressed protein Canstatin
3.Canstatin protein inhibits human hepatoma HepG2 cells value
4.Canstatin protein inhibits in vitro culture conditions in human hepatoma HepG2 cells the formation of vasculogenic mimicry
5.Canstatin protein inhibit in vitro culture conditions, HepG2 cells the expression of VE-cadherin protein, but no effect on the expression of MMp2.
引文
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