人脑胶质瘤中亲环素A蛋白的表达及临床意义
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摘要
胶质瘤是中枢神经系统最常见的恶性肿瘤,恶性程度高,早期诊断困难,根治机会较少,五年生存率低。最近研究示CypA在多数肿瘤组织中高度表达。本课题组以往研究结果证实缺氧可诱导肿瘤细胞CypA表达上调。目前研究已证实CypA与肿瘤的发生、发展、耐药及转移等相关,针对CypA的RNA干扰技术、CypA蛋白抑制剂能够抑制肿瘤细胞增殖、促进肿瘤细胞凋亡。提示CypA可作为治疗肿瘤的新靶点。CypA与肿瘤的研究已成为热点,但关于CypA和胶质瘤相关文献相对匮乏。故本课题组拟应用免疫组织化学方法观察CypA在胶质瘤中的表达与临床病理分级和预后之间的关系,拟采用RT-PCR、蛋白印迹等技术检测缺氧对C6细胞CypA表达的影响,拟利用小干扰RNA技术和MTT比色法检测抑制缺氧所诱导的CypA表达对C6细胞增殖的影响;为深入开展CypA和胶质瘤研究,拟利用基因工程技术表达GST-CypA融合蛋白,并优化融合蛋白纯化过程。
经研究证实人脑胶质瘤组织中CypA蛋白表达与胶质瘤临床病理分级、预后等因素密切相关,证实缺氧诱导C6细胞内CypA mRNA和蛋白表达水平上调,证实用小干扰RNA技术沉默缺氧诱导的C6细胞内CypA mRNA表达能够抑制C6细胞的增殖能力;成功构建pGEX-KG/CypA表达载体,并表达GST-CypA融合蛋白;用2%甲酸成功优化GST-CypA融合蛋白纯化过程,所纯化的融合蛋白具有CypA天然生物活性,为深入开展CypA和胶质瘤研究提供了保障。
本研究提示:CypA可成为一个胶质瘤临床辅助诊断的分子标志物,同时可成为胶质瘤治疗的新靶点。
Glioma is the most frequently occurring tumor in the central nervous system, occupying 40%-50% of the intracranial tumors. During the last twenty years, although there has been great advance in the clinical diagnosis and treatment of glioma, the prognosis of these patients has not improved obviously and their mortality is still high. The mean value of survival time is only 52 weeks in the patients with malignant glioma. Therefore, human brain glioma is still the formidable disorder for human to deal with among the neurosurgical tumors, and most researchers are focusing not only on its etiology, pathogenesis, biological characteristics but also the new and effective techniques for treatment.
Cyclophilin A (CypA), the prototypical member of the cyclophilin family, is a highly conserved protein in mammalian cells. It possesses enzymatic peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is essential for protein folding in vivo. Recently, it has been reported that CypA is overexpressing in many kinds of tumor cells, especially in solid tumors. These reports suggest that CypA might be the important factor for tumorigenesis in solid tumors. It is well known that most solid tumors contain hypoxic regions, and hypoxia affects a variety of tumor cell properties such as cell growth rate, neovascularization, metastasis and sensitivity to treatment. Hypoxia-inducible factor 1 alpha (HIF-1alpha), the main regulator of cellular hypoxic response in human tumor cells. HIF-1alpha is overexpressing in human gliomas and is a significant correlation between the tumor malignancy and invasiveness in human gliomas. In addition, our previous studies and other researches showed that overexpressing CypA in cancer cells might protect cells against cellular stresses, including hypoxia and cisplatin treatment, at least in part as a result of its antioxidant function.
Therefore, To The study of the relationship between CypA expression with tumor pathological grade and prognosis in human brain glioma. To investigate the changes of the expression of CypA in the cultured after conditioned hypoxia, we cultured C6 cells in hypoxia and examined the expression of CypA protein and mRNA by Western blot and RT-PCR. We used RNAi technique in vitro to suppress the expressing of CypA gene, the cell viability of each group was assayed by MTT colorimetry. The pGEX-KG-CypA recombinant vector was transformed successfully into E.coli. The highly purified GST-cyclophilin A (CypA) fusion protein was obtained. The procedure is also cost-effective, due to the low cost of 2% formic acid as compared with GSH.
This study can be described as follows:
1. The study of the relationship between CypA expression with tumor pathological grade and prognosis in human brain gliomas.
Objective: (1)To study relationship between the expression of CypA and the classification in human brain gliomas; (2)To investigate the relationship between the expression of CypA protein and prognosis in human brain gliomas. Methods: The expressions of CypA were determined by immunohistochemical technique in 45 specimens of gliomas. The relation between CypA expressions and prognosis of the patients with gliomas were analyzed. Forty five samples of gliomas were divided into 4 groups by the WHO standard, of the 45 cases, 6 cases were with pathological grade I, 10 with grade II, 13 with grade III, 16 with grade IV, 10 normal brain samples as controls. All the samples were stained with ABC immunohistochemistry, to observe the expression of CypA and the gliomas malignancy.
Results: (1)CypA positive expression rate was 0% in normal brain tissues and 60% in gliomas. There was significantly diference in the expression of CypA between the gliomas and normal brain tissues (P <0.01); (2)CypA positive expression rate was 16.7% in pathological Grade I, 40.0% in Grade II, 69.2% in Grade III, 81.3% in Grade IV. 31.3% in Grade I+II and 75.9% in Grade III+IV. CypA was more highly expressed in high grades than in low grades(P<0.01); (3)Strong positive expression of CypA was observed in 11 specimens of gliomas(24.4%), moderate in 7(15.6%), and weak in 9(20.0%), and negative expression was found in 18(40.0%). The data of following-up suggested that the survival time was significantly correlated with expression level of CypA(r=– 0.864, P <0.01); (4)The expression of CypA in recurrent astrocytoma group(90.9%) was higher that in primary astrocytoma group(50.0%)(P <0.05).
Conclusions: (1)There is no expression of CypA in normal cerebral cel1, CypA is over expressed in gliomas. CypA is a significant corelation with glioma grades; (2)The expressions of CypA protein were related to the prognosis of gliomas, and can be considered as the indicators of the prognosis. The present results suggest that CypA could be the molecular marker in clinic auxiliary diagnosis of glioma and it might be the potential target of glioma treatment.
2. The variation and its significance of the cyclophilin A expression in the C6 rat glioma cells by hypoxia.
Objective: To investigate the changes of the expression of CypA in the cultured after conditioned hypoxia.
Methods: We cultured C6 cells in hypoxia and examined the expression of CypA protein and mRNA by Western blot and RT-PCR. We used RNAi technique in vitro to suppress the expression of CypA gene, the cell viability of each group was assayed by MTT colorimetry.
Results: (1)The expression of CypA protein and mRNA increased after 1h and the highest level at 12 h under hypoxic conditions; (2)The expression level of CypA protein was significantly inhibited in C6 cells by CypA-siRNA, and the cell viability decreased singnificantly(P<0.01).
Conclusion: The present results suggerst that hypoxia might induce the increase of CypA expression in C6 cells, and the cell viability decrease significantly after inhibiting the expression of CypA. It implys that CypA gene may be one of the nover targets to inhibit growth of glioma cells.
3. Expression and Optimizing for purification of recombinant GST-cyclophilin A protein from E.coli.
Objective: To investigated a new method for purification of functional recombinant GST-cyclophilin A protein from E.coli, without affecting their structural and functional characteristics.
Methods: (1)Competent bacterium TOP10 was prepared with CaCl2. The plasmid pGEX-KG-CypA were transformed into E.coli host TOP10; (2)The elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer; (3)PPIase and chaperone activities assays were performed to determine whether the CypA part in the purified GST-CypA fusion protein maintained specific CypA functions.
Results: (1)The pGEX-KG-CypA recombinant vector was transformed successfully into E.coli host TOP10; (2)The molecular weight of GST-CypA fusion protein was 47KD; (3)The highly purified GST-CypA fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities.
Conclusion:(1)The GST-CypA fusion protein was efficiently expressed in E.coli; (2)By using this method, highly purified GST-CypA fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities; (3)The procedure is also cost-effective, due to the low cost of 2% formic acid as compared with GSH.
In summary, the results of this study show that the expression of CypA is closely related to the pathological grade as well as the prognosis of gliomas, which suggests that CypA might be the molecular marker in clinic auxiliary diagnosis of glioma and become the potential target in the treatment of glioma in the future. Secondly, the consequence of increasing of CypA expression in C6 cells under the circumstance of hypoxia implys that CypA might be related to the glioma resisting to hypoxia. Thirdly, the technique of RNAi in vitro can suppress the expression of CypA gene in C6 cells resulting from the situation of hypoxia, which implys that CypA gene may be one of the nover targets to inhibit growth of glioma cells. Fourthly, GST- CypA fusion protein was obtained from the transformation of the pGEX-KG-CypA recombinant vector and the former possesses the natural biological activity such as the activities of PPIase and chaperone, which suggests that the GST-CypA fusion protein might be applied to the research related to CypA and glioma. Last but not least, the utilization of the elution buffer containing 2% formic acid rather than GSH to elute the GST-fusion protein can obtain the highly purified GST-CypA fusion protein economically without changing the activity of the aim protein, which is in favor of obtaining highly purified and huge amounts of GST-CypA fusion proteins that can be implied to more researches related to CypA and glioma.
经研究证实人脑胶质瘤组织中CypA蛋白表达与胶质瘤临床病理分级、预后等因素密切相关,证实缺氧诱导C6细胞内CypA mRNA和蛋白表达水平上调,证实用小干扰RNA技术沉默缺氧诱导的C6细胞内CypA mRNA表达能够抑制C6细胞的增殖能力;成功构建pGEX-KG/CypA表达载体,并表达GST-CypA融合蛋白;用2%甲酸成功优化GST-CypA融合蛋白纯化过程,所纯化的融合蛋白具有CypA天然生物活性,为深入开展CypA和胶质瘤研究提供了保障。
本研究提示:CypA可成为一个胶质瘤临床辅助诊断的分子标志物,同时可成为胶质瘤治疗的新靶点。
Glioma is the most frequently occurring tumor in the central nervous system, occupying 40%-50% of the intracranial tumors. During the last twenty years, although there has been great advance in the clinical diagnosis and treatment of glioma, the prognosis of these patients has not improved obviously and their mortality is still high. The mean value of survival time is only 52 weeks in the patients with malignant glioma. Therefore, human brain glioma is still the formidable disorder for human to deal with among the neurosurgical tumors, and most researchers are focusing not only on its etiology, pathogenesis, biological characteristics but also the new and effective techniques for treatment.
Cyclophilin A (CypA), the prototypical member of the cyclophilin family, is a highly conserved protein in mammalian cells. It possesses enzymatic peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is essential for protein folding in vivo. Recently, it has been reported that CypA is overexpressing in many kinds of tumor cells, especially in solid tumors. These reports suggest that CypA might be the important factor for tumorigenesis in solid tumors. It is well known that most solid tumors contain hypoxic regions, and hypoxia affects a variety of tumor cell properties such as cell growth rate, neovascularization, metastasis and sensitivity to treatment. Hypoxia-inducible factor 1 alpha (HIF-1alpha), the main regulator of cellular hypoxic response in human tumor cells. HIF-1alpha is overexpressing in human gliomas and is a significant correlation between the tumor malignancy and invasiveness in human gliomas. In addition, our previous studies and other researches showed that overexpressing CypA in cancer cells might protect cells against cellular stresses, including hypoxia and cisplatin treatment, at least in part as a result of its antioxidant function.
Therefore, To The study of the relationship between CypA expression with tumor pathological grade and prognosis in human brain glioma. To investigate the changes of the expression of CypA in the cultured after conditioned hypoxia, we cultured C6 cells in hypoxia and examined the expression of CypA protein and mRNA by Western blot and RT-PCR. We used RNAi technique in vitro to suppress the expressing of CypA gene, the cell viability of each group was assayed by MTT colorimetry. The pGEX-KG-CypA recombinant vector was transformed successfully into E.coli. The highly purified GST-cyclophilin A (CypA) fusion protein was obtained. The procedure is also cost-effective, due to the low cost of 2% formic acid as compared with GSH.
This study can be described as follows:
1. The study of the relationship between CypA expression with tumor pathological grade and prognosis in human brain gliomas.
Objective: (1)To study relationship between the expression of CypA and the classification in human brain gliomas; (2)To investigate the relationship between the expression of CypA protein and prognosis in human brain gliomas. Methods: The expressions of CypA were determined by immunohistochemical technique in 45 specimens of gliomas. The relation between CypA expressions and prognosis of the patients with gliomas were analyzed. Forty five samples of gliomas were divided into 4 groups by the WHO standard, of the 45 cases, 6 cases were with pathological grade I, 10 with grade II, 13 with grade III, 16 with grade IV, 10 normal brain samples as controls. All the samples were stained with ABC immunohistochemistry, to observe the expression of CypA and the gliomas malignancy.
Results: (1)CypA positive expression rate was 0% in normal brain tissues and 60% in gliomas. There was significantly diference in the expression of CypA between the gliomas and normal brain tissues (P <0.01); (2)CypA positive expression rate was 16.7% in pathological Grade I, 40.0% in Grade II, 69.2% in Grade III, 81.3% in Grade IV. 31.3% in Grade I+II and 75.9% in Grade III+IV. CypA was more highly expressed in high grades than in low grades(P<0.01); (3)Strong positive expression of CypA was observed in 11 specimens of gliomas(24.4%), moderate in 7(15.6%), and weak in 9(20.0%), and negative expression was found in 18(40.0%). The data of following-up suggested that the survival time was significantly correlated with expression level of CypA(r=– 0.864, P <0.01); (4)The expression of CypA in recurrent astrocytoma group(90.9%) was higher that in primary astrocytoma group(50.0%)(P <0.05).
Conclusions: (1)There is no expression of CypA in normal cerebral cel1, CypA is over expressed in gliomas. CypA is a significant corelation with glioma grades; (2)The expressions of CypA protein were related to the prognosis of gliomas, and can be considered as the indicators of the prognosis. The present results suggest that CypA could be the molecular marker in clinic auxiliary diagnosis of glioma and it might be the potential target of glioma treatment.
2. The variation and its significance of the cyclophilin A expression in the C6 rat glioma cells by hypoxia.
Objective: To investigate the changes of the expression of CypA in the cultured after conditioned hypoxia.
Methods: We cultured C6 cells in hypoxia and examined the expression of CypA protein and mRNA by Western blot and RT-PCR. We used RNAi technique in vitro to suppress the expression of CypA gene, the cell viability of each group was assayed by MTT colorimetry.
Results: (1)The expression of CypA protein and mRNA increased after 1h and the highest level at 12 h under hypoxic conditions; (2)The expression level of CypA protein was significantly inhibited in C6 cells by CypA-siRNA, and the cell viability decreased singnificantly(P<0.01).
Conclusion: The present results suggerst that hypoxia might induce the increase of CypA expression in C6 cells, and the cell viability decrease significantly after inhibiting the expression of CypA. It implys that CypA gene may be one of the nover targets to inhibit growth of glioma cells.
3. Expression and Optimizing for purification of recombinant GST-cyclophilin A protein from E.coli.
Objective: To investigated a new method for purification of functional recombinant GST-cyclophilin A protein from E.coli, without affecting their structural and functional characteristics.
Methods: (1)Competent bacterium TOP10 was prepared with CaCl2. The plasmid pGEX-KG-CypA were transformed into E.coli host TOP10; (2)The elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer; (3)PPIase and chaperone activities assays were performed to determine whether the CypA part in the purified GST-CypA fusion protein maintained specific CypA functions.
Results: (1)The pGEX-KG-CypA recombinant vector was transformed successfully into E.coli host TOP10; (2)The molecular weight of GST-CypA fusion protein was 47KD; (3)The highly purified GST-CypA fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities.
Conclusion:(1)The GST-CypA fusion protein was efficiently expressed in E.coli; (2)By using this method, highly purified GST-CypA fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities; (3)The procedure is also cost-effective, due to the low cost of 2% formic acid as compared with GSH.
In summary, the results of this study show that the expression of CypA is closely related to the pathological grade as well as the prognosis of gliomas, which suggests that CypA might be the molecular marker in clinic auxiliary diagnosis of glioma and become the potential target in the treatment of glioma in the future. Secondly, the consequence of increasing of CypA expression in C6 cells under the circumstance of hypoxia implys that CypA might be related to the glioma resisting to hypoxia. Thirdly, the technique of RNAi in vitro can suppress the expression of CypA gene in C6 cells resulting from the situation of hypoxia, which implys that CypA gene may be one of the nover targets to inhibit growth of glioma cells. Fourthly, GST- CypA fusion protein was obtained from the transformation of the pGEX-KG-CypA recombinant vector and the former possesses the natural biological activity such as the activities of PPIase and chaperone, which suggests that the GST-CypA fusion protein might be applied to the research related to CypA and glioma. Last but not least, the utilization of the elution buffer containing 2% formic acid rather than GSH to elute the GST-fusion protein can obtain the highly purified GST-CypA fusion protein economically without changing the activity of the aim protein, which is in favor of obtaining highly purified and huge amounts of GST-CypA fusion proteins that can be implied to more researches related to CypA and glioma.
引文
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