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目的构建人组织因子途径抑制物2(TFPI2)基因的原核表达载体并在大肠杆菌中有效表达;探索重组TFPI2抑制纤溶酶活性及其对人卵巢癌细胞(A2780)迁徙浸润能力的影响。方法1.以TFPI2cDNA为模板,PCR扩增得到645bpDNA片段,克隆入表达载体pET28a,并转化大肠杆菌BL21株表达。2.限制性内切酶和菌落PCR法鉴定TFPI2基因DNA片段,Westernblot法鉴定TFPI2融合蛋白。3.建立TFPI2抑制纤溶酶的动力学方法并测定重组TFPI2对纤溶酶的抑制作用。4.在体外用Boyden小室,以不同浓度TFPI2作用A2780细胞进行迁徙和浸润实验。结果1.成功构建重组TFPI2的原核表达载体pET28a并在BL21株中表达、纯化,获得大量TFPI2蛋白。2.Westernblot证实表达的融合蛋白为TFPI2。3.重组TFPI2对液相和固相中的纤溶酶具有显著抑制作用,且呈剂量效应关系。4.在迁徙实验中,将A2780TFPI2不同浓度组与A2780对照组细胞穿过人工膜的细胞数进行比较,经t检验,无统计学意义(P>0.05);在浸润实验中,将A2780TFPI2不同浓度组与A2780对照组细胞穿过人工膜的细胞数进行比较及TFPI2不同浓度组间的细胞数进行比较,经t检验,具有显著差异(P<0.01)。结论1.人TFPI2基因的表达,为TFPI2病理生理的作用和临床研究提供了丰富材料。2.重组TFPI2抑制纤溶酶活性的研究,为探讨TFPI2对人卵巢癌细胞浸润转移能力的影响等提供了实验依据。3.重组TFPI2对人卵巢癌细胞体外自身运动能力无影响,但可显著抑制人卵巢癌细胞的体外浸润能力,为将来卵巢癌用蛋白酶抑制剂治疗提供一可能的靶向依据。
Objective To construct prokaryotic expression vector of human TFPI2 gene and express in Escherichia coli efficiently. To explore the effect of recombinant TFPI2 on plasmin activity and migration and invasion of human ovarian cancer cell line A2780. Method 1. Using TFPI2 cDNA as a template, a 645bp DNA fragment was amplified by PCR, cloned into the expression vector pET28a and transformed into E. coli BL21. The DNA fragment of TFPI2 gene was identified by restriction endonuclease and colony PCR, and TFPI2 fusion protein was identified by Western blot. To establish a kinetic method for the inhibition of plasmin by TFPI2 and to determine the inhibitory effect of recombinant TFPI2 on plasmin. In vitro Boyden chamber, with different concentrations of TFPI2 A2780 cells migration and invasion experiments. The prokaryotic expression vector pET28a of recombinant TFPI2 was successfully constructed and expressed in BL21 strain, and a large amount of TFPI2 protein was obtained. Western Blot showed that the expressed fusion protein was TFPI2.3.The recombinant TFPI2 had a significant inhibitory effect on plasmin in liquid and solid phase, and showed a dose-response relationship. In the migratory experiment, comparing the cell numbers of cells in different concentrations of A2780TFPI2 with that of A2780 control group passing through the artificial membrane, there was no statistical significance by t test (P> 0.05); In the infiltration experiment, different concentrations of A2780TFPI2 The number of cells passing through the artificial membrane in A2780 control group and the number of cells in different concentrations of TFPI2 were compared. There was significant difference (P <0.01) by t test. Conclusion 1. The expression of human TFPI2 gene provides abundant materials for the role of TFPI2 in pathophysiology and clinical research. Recombinant TFPI2 inhibits the activity of plasmin, providing experimental evidence for exploring the effect of TFPI2 on the invasion and metastasis of human ovarian cancer cells. Recombinant TFPI2 has no effect on the self-motility of human ovarian cancer cells in vitro, but it can significantly inhibit the ability of human ovarian cancer cells to infiltrate in vitro and provide a possible target for future treatment of ovarian cancer with protease inhibitors.