【作者】 孙鸣宇；

【导师】 韩雅玲；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 硕士

【摘要】 目的E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes, CREG)是1998年Gill博士利用酵母双杂交技术克隆的一个转录调控相关因子。CREG基因可与外源性腺病毒蛋白E1A及哺乳动物体内转录因子E2F家族蛋白竞争性地结合在靶基因的启动子区,从而阻遏E1A蛋白和E2F对靶基因的转录,而E1A、E2F均具有促进细胞增殖的作用,所以CREG可能通过与E1A、E2F竞争性抑制作用而起到促进细胞分化、抑制细胞增殖的作用。本实验室前期研究发现,去血清能够诱导体外培养的人血管平滑肌细胞(vascular smooth muscle cells,VSMCs)从合成表型逆转为分化表型,同时CREG蛋白表达明显增加。大鼠颈动脉拉伤实验证实,CREG基因表达与VSMCs增殖能力呈负相关,推测其可能参与了VSMCs表型调控。Veal博士等证实CREG是一种分泌型糖蛋白,可以与胰岛素样生长因子II竞争性结合细胞膜胰岛素样生长因子受体II,以自分泌和旁分泌两种途径对细胞功能进行调控。为进一步明确CREG糖蛋白的功能,本研究采用逆转录-聚合酶链反应(RT-PCR)、蛋白印迹(Western blot)及Ni-NTA亲和层析等方法表达、纯化重组hCREG糖蛋白,并用流式细胞周期分析、5-溴脱氧尿嘧啶(BrdU)掺入实验等方法验证重组人CREG(hCREG)糖蛋白具备抑制体外培养的人胸廓内动脉平滑肌细胞(Human internal thoracic artery-Shenyang, HITASY)增殖的生物学功能,为hCREG蛋白的工程化生产提供前期研究基础。方法①用RT-PCR技术扩增终止密码突变的hCREG开放读码框并构建pcDNA3.1 myc-His/hCREG真核表达载体。②Lipofectamine 2000转染人293F细胞株并筛选稳定表达细胞克隆,Western blot方法鉴定hCREG/myc-His融合蛋白的表达,糖苷酶法及Western blotting行hCREG/myc-His融合蛋白的糖基化分析。③根据6×His亲和层析原理,应用Ni-NTA柱纯化重组hCREG蛋白,纯化后蛋白通过HiTrap脱盐柱脱盐。④用流式细胞周期分析研究添加0.5μg /mL、1μg /mL及2μg /mL重组hCREG/myc-His融合糖蛋白对体外培养HITASY细胞增殖的影响。用BrdU掺入方法研究添加重组蛋白对体外培养HITASY细胞增殖的影响。结果RT-PCR扩增含终止密码突变的hCREG cDNA片段,经BamH I和EcoR I双酶切构建了pcDNA 3.1myc-His/hCREG真核表达载体,酶切及测序结果均证实构建的重组质粒正确。稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞及未转染的对照细胞裂解产物,分别以Anti-hCREG、Anti-myc及Anti-His行Western blot检测。hCREG抗体可检测到转染组较对照组多两条约30KD的融合蛋白表达条带,myc抗体及His抗体均检测到大小约为30KD的融合蛋白表达。将稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞裂解产物及经PNGaseF处理过的样品分别行Anti-hCREG、Anti-myc及Anti-His的Western blot检测。结果显示经PNGaseF处理后,hCREG融合蛋白分子量由30KD降低至25KD,电泳条带下移,证明带有myc和His标签的重组hCREG蛋白为糖基化蛋白。根据6×His与Ni亲合层析的原理用Ni-NTA纯化hCREG重组蛋白。收集的洗脱液经Centriprep离心超滤管(10000NMWL)浓缩后,经BCA法测定并与蛋白标准曲线比较,重组hCREG蛋白浓度为1.6 mg/mL。考马斯亮兰染色可见30KD大小的较单一条带,经image-J软件分析,纯度为92%。浓缩并脱盐后的纯化蛋白经PNGaseF处理后分子量由30KD降至25KD,条带下移,证实纯化后的蛋白仍保留了糖基化形式。流式细胞仪细胞周期分析结果提示重组hCREG蛋白与对照组相比可抑制体外培养的HITASY细胞增殖,G0/G1期细胞比率(共计数2万个细胞)显著增加,且低浓度组的抑制效应要高于高浓度组。BrdU掺入实验结果提示,添加2μg /mL重组hCREG蛋白组与对照组相比可显著抑制体外培养的HITASY细胞增殖,BrdU阳性细胞的比率降低。组间比较有统计学差异(P<0.05)。结论pcDNA 3.1 myc-His/hCREG真核表达载体的构建及具有生物学功能的重组hCREG/myc-His糖蛋白的表达纯化,为hCREG蛋白的功能研究及生物工程制备提供了实验平台。纯化的重组hCREG蛋白可能作为一种新的药物涂于支架表面,通过其促进分化、抑制增殖的生物学功能实现降低支架内再狭窄的作用。更多还原

【Abstract】 Objective The cellular repressor of E1A- stimulated genes (CREG), as a transcriptional regulator, was cloned by the yeast two-hybrid method by Dr. Gill in 1998. The result that CREG could inhibit the transcription of target gene by E1A protein and E2F may contribute to the binding of CREG competing with the exogenous adenovirus E1A protein and transcription factor of E2F family proteins in mammals to the promoter region of target genes. Both E1A protein and E2F can promote cellular dedifferentiation and proliferation, CREG is therefore likely to play a role in promoting differentiation and/or inhibiting cell growth by competing with E1A and E2F.Our early research had found that the expression of CREG synchronously increased when the human vascular smooth muscle cells (VSMCs) cultured in vitro converted from synthetic into differentiated phenotype induced by serum withdrawal. The negative relationship between CREG mRNA expression and the proliferative activity of VSMCs was confirmed in rat injury carotid artery. CREG, as a secreted glycoprotein reported by Dr. Veal, could bind to the transmembrane insulin-like growth factor II receptor competing with the insulin-like growth factor II and regulate cell function through both autocrine and paracrine pathway. To study the bio-function of CREG glycoprotein, in this study, the reverse transcriptionpolymerase chain reaction(RT-PCR), Western blot and affinity chromatograph by Ni-NTA were used to express and purify the recombinant hCREG/myc-his glycoprotein. Flow cytometic analysis and BrdU incorporation method were used to confirm the biological function of hCREG/myc-his which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro. The work provided prophase research basis for engineering production of hCREG protein.Methods①The ORF(open reading frame) fragment of human CREG(hCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector pcDNA3.1 myc-His/hCREG was constructed.②The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000, and stably transfected cell clones were screened by G418. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot.③The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. The recombinant hCREG/ myc-His protein was concentrated and desalted. The concentration of recombinant protein was analyzed by SDS-PAGE and the purity was calculated by bio-informational software.④ - 8 -The effect of recombinant hCREG/myc-His glycoprotein of different concentration(0.5μg /mL、1μg /mL and 2μg /mL)on proliferation of HITASY cells was confirmed by Flow cytometic analysis and BrdU incorporation method.Results The ORF fragment of hCREG was amplificated by RT-PCR and inserted into the pcDNA3.1 myc-His vector with EcoRΙ/BamHΙdigestion. The recombinant eukaryotic expression vector (pcDNA3.1 myc-His/ hCREG) was confirmed to be constructed successfully by restricted endonuclease digestion and DNA sequencing.The lysates of 293F cells with stably transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant fusion protein in cell lysates was identified as a glycoprotein because the molecular weight of fusion protein decreased from 30KD to 25KD after PNGaseF digestion. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices(10000NMWL), the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Glycoprotein was identified using PNGaseF digestion.The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro by Flow cytometic analysis and BrdU incorporation method. The inhibition effect of recombinant hCREG protein is stronger in low-dosage treatment than in high-dosage group. There was statistical difference among the groups(P<0.05). Conclusion The construction of eukaryotic expression vector of pcDNA3.1 myc-His/hCREG and the expression and purification of recombinant hCREG/myc-His glycoprotein with biological activity provided an experiment platform for function study and engineering production of hCREG protein. The incidence of restenosis may be reduced if the stents were coated with recombinant hCREG/myc-His glycoprotein.更多还原