一种新的抗辐射性相关蛋白AAl2和CHKl相互作用研究

目的：研究AAl2蛋白与CHKl蛋白的相互作用。方法：用在大肠杆菌中表达纯化的AAl2(LG21)蛋白制备多克隆抗体，Western印迹检测抗体的特异性；用自制的抗体在A1-5和B4细胞中进行了免疫共沉淀实验；同时将AAl2与Chkl基因克隆入酵母双杂交载体中，将重组质粒导入酵母菌AH109中，检测报告基因的表达情况。结果：Western印迹结果表明该抗体可以与AAl2蛋白特异结合；并用免疫共沉淀实验验证了从12蛋白与CHKl蛋白在哺乳动物细胞体内的相互作用。同时用酵母双杂交实验验证了AAl2蛋白与CHKl蛋白在酵母体内的相互作用，β-半乳糖苷酶活性分析和α-半乳糖苷酶活性分析结果均为阳性。结论：AAl2蛋白和CHKI蛋白之间存在相互作用，此结果为进一步研究新基因AAl2的功能奠定了基础。     

非受体酪氨酸激酶c-Abl与Siva-1相互作用及在细胞凋亡中的功能研究

目的：研究非受体酪氨酸激酶c-Abl与Siva-1蛋白的相互作用及其对细胞凋亡的影响。方法：应用免疫共沉淀研究蛋白质相互作用；抗酪氨酸抗体研究蛋白体内磷酸化；体外激酶分析研究体外磷酸化；流式细胞仪测定Sub G1细胞百分比以测定细胞调亡。结果：非受体酪氨酸激酶c-Abl在细胞内及体外均与Siva-1形成复合物，通过体外翻译的c-Abl结合实验表明其为直接的相互作用，其相互作用主要通过c-Abl的SH3结构域与Siva-1直接结合，且Siva-1可以被c-Abl磷酸化。通过c-Abl显性负突变结果表明，酪氨酸磷酸化对Siva-1诱导细胞凋亡至关重要。结论：c-Abl使Siva-1磷酸化可促进Siva-1的细胞凋亡功能。     

Mpl相互作用蛋白RACK1的筛选与鉴定

目的：筛选新的血小板生成素(TPO)受体胞内区相互作用蛋白并确定相互作用。方法：以TPO受体胞内区为诱饵蛋白，进行酵母双杂交文库的筛选，RT-PCR扩增筛选到的基因并克隆到相应栽体上，Westem印迹方法检测其在不同组织与细胞内的表达；进行哺乳动物细胞内双杂交、免疫共沉淀、激光共聚焦实验确证相互作用。结果：从酵母双杂交文库筛选到RACK1基因，检测到其在不同组织与细胞内表达都很丰富，哺乳动物细胞内双杂交、免疫共沉淀证明RACK1同TPP受体Mpl的胞内区存在相互作用，而细胞内共定位实验证明两者在哺乳动物细胞内存在共定位现象，即两蛋白的相互作用可能具有生理意义。结论：筛选到的RACK1蛋白同TPO受体Mpl的胞内区存在相互作用。     

Tat蛋白与Plk1相互作用位点的鉴定

目的确定Tat蛋白与Plk1的相互作用及作用位点。方法构建tat和plk1基因的不同表达载体,通过GST-pull down、免疫共沉淀检测它们表达产物的相互作用位点,激光共聚焦确定其在细胞内的定位。结果 GST-pull down和免疫共沉淀实验发现Tat蛋白能结合在Plk1 N端的激酶区域,激光共聚焦发现Tat蛋白和Plk1在细胞核内有相同的亚细胞定位。结论 Tat蛋白能结合在Plk1 N端的激酶区域引起Plk1 T210的磷酸化水平提高,从而激活Plk1的活性,导致细胞周期的紊乱。     

一种新的抗辐射性相关蛋白AA12和CHK1相互作用研究

目的研究AA12蛋白与CHK1蛋白的相互作用.方法用在大肠杆菌中表达纯化的AA12(LG21)蛋白制备多克隆抗体,Western印迹检测抗体的特异性;用自制的抗体在A1-5和B4细胞中进行了免疫共沉淀实验;同时将AA12与Chk1基因克隆入酵母双杂交载体中,将重组质粒导入酵母菌AH109中,检测报告基因的表达情况.结果Western印迹结果表明该抗体可以与AA12蛋白特异结合;并用免疫共沉淀实验验证了AA12蛋白与CHK1蛋白在哺乳动物细胞体内的相互作用.同时用酵母双杂交实验验证了AA12蛋白与CHK1蛋白在酵母体内的相互作用,β-半乳糖苷酶活性分析和α-半乳糖苷酶活性分析结果均为阳性.结论AA12蛋白和CHK1蛋白之间存在相互作用,此结果为进一步研究新基因AA12的功能奠定了基础.     

人CDK7基因真核表达载体的构建与鉴定

目的：构建带Myc标签的细胞周期蛋白依赖性激酶7（cyclin－dependent kinase 7, CDK7）的真核表达载体,获得其表达产物,并验证该激酶与已知相互作用蛋白P53之间的相互作用。方法应用PCR技术从人乳腺文库中扩增出CDK7全长编码区基因,将其克隆到pXJ－40载体中;继而重组质粒转染人胚肾293 T细胞,以SDS－PAGE和Western印迹鉴定表达情况;免疫共沉淀检测Myc－CDK7与FLAG－p53的相互作用。结果双酶切和基因测序鉴定显示,Myc－CDK7真核表达质粒克隆构建成功;SDS－PAGE和Western印迹结果表明,Myc－CDK7转染人胚肾293T细胞后成功表达;免疫共沉淀显示,重组Myc－CDK7与已知相互作用蛋白P53在蛋白质水平上具有相互作用,证实其具有生物学活性。结论成功构建Myc－CDK7的真核表达载体,为进一步探讨Myc－CDK7对细胞周期的调控奠定了实验基础。     

PTD-OD-HA融合蛋白对K562细胞中Bcr-Ab1的抑制作用观察

目的 探讨PTD-OD-HA融合蛋白转导K562细胞的动力学、定位及其与Bcr-Ab1定位的关系,以及对Bcr-Ab1寡聚化和Bcr-Ab1酪氨酸激酶活性的影响.方法 采用FITC标记PTD-OD-HA融合蛋白,观察蛋白转导K562细胞的效率与剂量和时间的关系,激光共聚焦显微镜检测融合蛋白在细胞内的定位,免疫共沉淀法检测融合蛋白与Bcr-Ab1的相互作用,Western blotting检测Bcr-Ab1的磷酸化水平.结果 PTD-OD-HA转导K562细胞呈剂量和时间依赖性.PTD-OD-HA定位于K562细胞胞质,与Bcr-Ab1共定位,且能与Bcr-Ab1蛋白相互作用,进而干扰Bcr-Ab1同源寡聚化,并可降低Bcr-Ab1的磷酸化水平.结论 在K562细胞中,PTD-OD-HA融合蛋白能有效抑制Bcr-Ab1同源寡聚化及磷酸化水平.     

PTD-OD-HA融合蛋白对K562细胞中Bcr-Ab1的抑制作用观察

目的探讨PTD-OD-HA融合蛋白转导K562细胞的动力学、定位及其与Bcr-Abl定位的关系,以及对Bcr-Abl寡聚化和Bcr-Abl酪氨酸激酶活性的影响。方法采用FITC标记PTD-OD-HA融合蛋白,观察蛋白转导K562细胞的效率与剂量和时间的关系,激光共聚焦显微镜检测融合蛋白在细胞内的定位,免疫共沉淀法检测融合蛋白与Bcr-Abl的相互作用,Western blotting检测Bcr-Abl的磷酸化水平。结果 PTD-OD-HA转导K562细胞呈剂量和时间依赖性。PTD-OD-HA定位于K562细胞胞质,与Bcr-Abl共定位,且能与Bcr-Abl蛋白相互作用,进而干扰Bcr-Abl同源寡聚化,并可降低Bcr-Abl的磷酸化水平。结论在K562细胞中,PTD-OD-HA融合蛋白能有效抑制Bcr-Abl同源寡聚化及磷酸化水平。     

东部马脑炎病毒E2基因的表达及DNA免疫的初步研究

目的：构建东部马脑炎病毒E2基因的重组真核表达载体，对其DNA免疫原性进行观察。方法：采用RT-PCR扩增东部马脑炎病毒全长E2基因，构建真核表达载体pcDNA-E2，以脂质体法转染COS7细胞，经蛋白印迹法和免疫荧光法证明E2基因可以表达后，用庐重组质粒DNA免疫Balb/c小鼠，并用免疫荧光法检测鼠血清中病毒的特异抗体。结果：免疫印迹法、免疫荧光法检测表明E2基因在COS7细胞中获得瞬时表达，pcDNA-E2基因免疫小鼠可产生抗东部脑炎病毒的特异抗体。结论：东部马脑炎病毒E2基因的重组质粒DNA可刺激小鼠产生特异性的抗东部马脑炎病毒体液免疫应答，为基因疫苗的研制奠定了基础。     

分化抑制因子1促进血管内皮细胞增殖的机制研究

目的探讨分化抑制因子1(Id1)促进血管内皮细胞增殖的机制。方法构建p21启动子介导的报告基因p21-Luc,分别转染Eahy926细胞、Id1抑制(siId1细胞)或诱导表达(pcDNAId1)细胞、E2A抑制或诱导表达细胞,检测荧光素酶报告基因活性。提取Eahy926细胞总蛋白,分别加入Id1抗体、E12抗体和E47抗体,免疫共沉淀,以Eahy926细胞作为对照,Western blotting检测Id1、E12和E47的表达情况。分别转染siId1、pcDNAId1载体于Eahy926细胞,与正常Eahy926细胞同步化培养48h后,行细胞染色,用流式细胞仪检测细胞G1期的DNA含量。结果与Eahy926细胞比较,siId1细胞、pcDNAE2A细胞p21基因启动子介导的荧光素酶活性增强,p21蛋白表达增高(P<0.05,P<0.01);pcDNAId1细胞、siE2A细胞p21基因启动子荧光素酶活性降低,p21蛋白表达减弱(P<0.05)。免疫共沉淀结果显示,Id1免疫共沉淀细胞裂解物中同时检测到E47/E12蛋白,E47/E12沉淀物中也能检测到Id1。与正常Eahy926细胞比较,转染siId1的...     

ATM参与辐射损伤细胞学反应的研究

目的 克隆ＡＴＭ全长ｃＤＮＡ及含特异功能域的ｃＤＮＡ片段,寻找与ＡＴＭ相互作用的蛋白,分析ＡＴＭ在ＤＮＡ损伤名的分子机理。方法 利用长片段ＰＣ白话 增法,从人外周血来源的ｃＤＮＡ库中扩增ＡＴＭｃＤＮＡ;利用酵母双杂交系统筛选人外周血来源的ｃＤＮＡ库中与ＡＴＭ相互作用的蛋白。结果 经重叠ＰＣＲ扩增到ＡＴＭ全长ｃＤＮＡ,并筛选到数条与ＡＴＭＰ１３Ｋ激酶区相互作用的ｃＤＮＡ,并对其序列进行分析。结果 Ａ     

中国人甲状腺乳头状癌中ret基因重排及类型的分析研究

目的 建立ret基因重排的分析方法，提示中国人甲状腺乳头状癌中是否存在原癌基因ret重排及重排类型。方法 应用RT—PCR方法对65例甲状腺乳头状癌石蜡切片标本进行ret基因重排检测，并对扩增结果进行测序鉴定。结果 在提取RNA成功的38例甲状腺乳头状癌(PK)标本中，有71％发生ret重排，其中10．5％表达RTC1；5．3％表达PTC3；5．3％表达PTC4，以PTC1发生的频率较高。仅1例表达PTC2与PTC3／PTC4混合型重排，中国人PTC可能与ret和H4及ELE1重排激活相关。结论 中国人PTC组织中存在4种重排，且以PTC1较为常见。单一标本中有多种重排形式并存，PTC1 3重排形式10例、3例PTC1 4、1例PTC2 3 4、1例PTC1 4、4例PTC1 3 4，单一标本中多种重排形式并存的比例高达50％，但与癌症的恶性程度、转移等无必然联系。     

Cleavage of ATM during radiation-induced apoptosis: caspase-3-like apoptotic protease as a candidate

Purpose : To study the relationship of ionizing radiation-induced apoptosis with the integrity of ATM (mutated in ataxia telangiectasia) that has a critical role in DNA damage sensing and repair, cell-cycle checkpoint controls and maintenance of genomic stability. Materials and methods : U937 cells were treated with gamma-radiation. Sub-G1 DNA content, DNA fragmentation, cleavage of PARP, active caspase-3, cleavage of ATM in vivo and in vitro were measured by flow cytometry, agarose gel electrophoresis, cleaving of colorimetric caspase-3 substrate and Western blotting. Results : ATM is specifically cleaved in cells during the induction of apoptosis by ionizing radiation exposure. The time-course of cleavage coincided with the appearance of cells with a sub-G1 DNA content and activation of caspase-3. ATM was cleaved with similar kinetics as PARP and DEVD-FMK could abolish the cleavage. In vitro studies showed that ATM was cleaved by caspase-3 or related subfamily members at a DIVD/G site. Conclusion : ATM belongs to a group of repair proteins, including PARP, DNA-PK and HsRad51, which are specifically cleaved during apoptosis. These findings support the idea that repair mechanisms need to be inactivated for apoptosis to proceed effciently.     

ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia.

PURPOSE: To provide an update on the product of the ATM gene mutated in the human genetic disorder ataxia-telangiectasia (A-T). SUMMARY: The product of the ATM gene mutated in the human genetic disorder A-T is a 350 kDa protein that plays a central role in the regulation of a number of cellular processes. It is a member of the phosphatidylinositol 3-kinase superfamily, but is more likely a protein kinase similar to another member of that family, i.e. DNA-dependent protein kinase (DNA-PK). A-T cells and fibroblasts derived from the atm -/- mouse are hypersensitive to ionizing radiation and defective in cell cycle checkpoint control. At present the nature of the lesion in damaged DNA recognized by ATM remains uncertain, but it is evident that a small number of residual strand breaks remain unrepaired in A-T cells, which may well account for the radiosensitivity. On the other hand, considerable progress has been achieved in delineating the role of ATM in cell cycle checkpoint control. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation. At the G1 /S interface ATM has been shown to play a central role in radiation-induced activation of the tumour suppressor gene product p53. ATM binds to p53 in a complex fashion and activates the molecule in response to breaks in DNA by phosphorylating it at serine 15 close to the N-terminus and by controlling other phosphorylation and dephosphorylation changes on the molecule. This in turn leads to the induction of p21/WAF1 and other p53 effector proteins before inhibition of cyclin-dependent kinase activity and G1 arrest. Emerging evidence supports a direct role for ATM at other cell cycle checkpoints. Other proteins interacting with ATM include c-Abl a protein tyrosine kinase, beta-adaptin an endosomal protein and p21 a downstream effector of p53. The significance of these interactions is currently being investigated. ATM also plays an important role in the regulation and surveillance of meiotic progression. The localization of ATM to both the nucleus and other subcellular organelles implicates this molecule in a myriad of cellular processes. CONCLUSION: ATM is involved in DNA damage recognition and cell cycle control in response to ionizing radiation damage. There is evidence that ATM may also have a more general signalling role.     

Cleavage of ATM during radiation-induced apoptosis: caspase-3-like apoptotic protease as a candidate

PURPOSE: To study the relationship of ionizing radiation-induced apoptosis with the integrity of ATM (mutated in ataxia telangiectasia) that has a critical role in DNA damage sensing and repair, cell-cycle checkpoint controls and maintenance of genomic stability. MATERIALS AND METHODS: U937 cells were treated with gamma-radiation. Sub-G1 DNA content, DNA fragmentation, cleavage of PARP, active caspase-3, cleavage of ATM in vivo and in vitro were measured by flow cytometry, agarose gel electrophoresis, cleaving of colorimetric caspase-3 substrate and Western blotting. RESULTS: ATM is specifically cleaved in cells during the induction of apoptosis by ionizing radiation exposure. The time-course of cleavage coincided with the appearance of cells with a sub-G1 DNA content and activation of caspase-3. ATM was cleaved with similar kinetics as PARP and DEVD-FMK could abolish the cleavage. In vitro studies showed that ATM was cleaved by caspase-3 or related subfamily members at a DIVD/G site. CONCLUSION: ATM belongs to a group of repair proteins, including PARP, DNA-PK and HsRad51, which are specifically cleaved during apoptosis. These findings support the idea that repair mechanisms need to be inactivated for apoptosis to proceed efficiently.     

Review: ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia

Purpose: To provide an update on the product of the ATM gene mutated in the human genetic disorder ataxia-telangiectasia (A-T). Summary : The product of the ATM gene mutated in the human genetic disorder A-T is a 350kDa protein that plays a central role in the regulation of a number of cellular processes. It is a member of the phosphatidylinositol 3-kinase superfamily, but is more likely a protein kinase similar to another member of that family, i.e. DNA-dependent protein kinase (DNA-PK). A-T cells and fibroblasts derived from the atm /- mouse are hypersensitive to ionizing radiation and defective in cell cycle checkpoint control. At present the nature of the lesion in damaged DNA recognized by ATM remains uncertain, but it is evident that a small number of residual strand breaks remain unrepaired in A-T cells, which may well account for the radiosensitivity. On the other hand, considerable progress has been achieved in delineating the role of ATM in cell cycle checkpoint control. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation. At the G1/S interface ATM has been shown to play a central role in radiation-induced activation of the tumour suppressor gene product p53. ATM binds to p53 in a complex fashion and activates the molecule in response to breaks in DNA by phosphorylating it at serine 15 close to the N-terminus and by controlling other phosphorylation and dephosphorylation changes on the molecule. This in turn leads to the induction of p21/WAF1 and other p53 effector proteins before inhibition of cyclin-dependent kinase activity and G1 arrest. Emerging evidence supports a direct role for ATM at other cell cycle checkpoints. Other proteins interacting with ATM include c-Abl a protein tyrosine kinase, beta -adaptin an endosomal protein and p21 a downstream effector of p53. The significance of these interactions is currently being investigated. ATM also plays an important role in the regulation and surveillance of meiotic progression. The localization of ATM to both the nucleus and other subcellular organelles implicates this molecule in a myriad of cellular processes. Conclusion: ATM is involved in DNA damage recognition and cell cycle control in response to ionizing radiation damage. There is evidence that ATM may also have a more general signalling role.     

Radiosensitization of tumor cells by modulation of ATM kinase

PURPOSE: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization. METHOD: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied. RESULTS: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts. CONCLUSIONS: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.     

Radiosensitization of tumor cells by modulation of ATM kinase

Purpose: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization.

Method: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied.

Results: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts.

Conclusions: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.