ATM基因辐射防护功能研究

[目的]研究ATM基因对毛细血管扩张性共济失调症(ataxia-telangiectasia,AT)患者皮肤成纤维细胞系AT5BIVA(AT细胞)高辐射敏感性的纠正情况,以评价ATM基因的辐射防护功能。[方法]利用电穿孔技术,将含有ATM基因cDNA的真核表达载体PEBS7-YZ5转染到AT细胞,用潮霉素筛选以获得稳定表达细胞株,RT-PCR检测ATMcDNA的转录以进一步验证;用常规染色体畸变分析法,在PEBS7-YZ5-AT细胞、PEBS7-AT细胞和未转染AT细胞经60Coγ射线0、1、2、3、4Gy照射后,观察比较三者间染色体畸变率(CAF)的差异。[结果]PEBS7-YZ5成功转进AT细胞,RT-PCR检测到ATMcDNA片段,在0、1、2、3、4Gy剂量下,PEBS7-YZ5-AT细胞染色体畸变率明显低于AT细胞,其差别具有显著性(P<0.05);而PEBS7-AT细胞和AT细胞间无明显差别(P>0.05),三种细胞染色体畸变率随受照剂量增加而升高。[结论]毛细血管扩张性共济失调症患者AT细胞高辐射敏感性被ATM基因纠正,ATM基因具有辐射防护功能,有望成为辐射防护剂。     

ATM与辐射激活的磷酸化P53、P21蛋白的相互作用

背景与目的:ATM基因属于PI-3K激酶家族成员,其编码蛋白具有调控DNA修复过程和调整细胞周期关卡的功能。毛细血管扩张性共济失调症(ataxia-telangiectasia,AT)患者AT细胞中ATM基因的突变导致了辐射诱发的P53、P21磷酸化缺失,说明辐射激活ATM基因可调控P53、P21的磷酸化。本实验利用免疫共沉淀及Westernblot技术来研究辐射激活的ATM基因与p53的关系,并观察ATM基因是否不通过P53而直接调控P21的磷酸化。方法:利用电穿孔技术将含有ATM基因cDNA的真核表达载体pEBS7-YZ5转染到AT细胞中,用潮霉素筛选以获得稳定表达细胞株,RT-PCR检测ATMcDNA的转录以进一步验证;在ATM稳定表达的AT细胞中,利用免疫共沉淀及Westernblot技术研究ATM基因与p53基因的相互关系;以K562细胞(p53突变)为p53突变细胞模型,研究ATM是否直接磷酸化P21。结果:pEBS7-YZ5成功转进AT细胞,RT-PCR检测到ATMcDNA片段;ATM稳定表达的AT细胞株在电离辐射诱导下,P53被磷酸化,免疫共沉淀显示ATM与P53相互作用;K562细胞经60Coγ射线照射后,P21被磷酸化,ATM抗体免疫共沉淀物中检测到P21蛋白的存在。结论:细胞遭受电离辐射作用后所激活的ATM激酶,可通过磷酸化P53继而活化细胞周期检控点P21蛋白,也可在电离辐射导致DNA损伤早期直接磷酸化P21蛋白,来启动DNA修复机制。     

人α1抗胰蛋白酶基因的克隆及在COS-7细胞中的表达

目的克隆人α1抗胰蛋白酶(α1-AT)基因并在真核细胞中表达。方法以人α1-抗胰蛋白酶(α1-AT)基因的总RNA为模板，采用反转录PCR法得到α1-AT的编码区cDNA，构建真核表达载体pcDNA3．1( )-α1-AT，在脂质体介导下转染非洲绿猴肾细胞系COS-7，经G418压力筛选建立稳定转染人α1-AT的细胞系，用Western印迹检测其在COS-7细胞中的表达。结果获得的人α1-AT cDNA序列与文献报道的核苷酸序列一致，Western印迹证实稳定转染人α1-AT的COS-7细胞系中有α1-AT的表达。结论构建了人α1抗胰蛋白酶的真核表达载体，并在COS-7细胞中获得稳定表达。     

ATM对辐射后AT细胞端粒酶活性的影响

目的:探讨ATM对电离辐射照射的毛细血管扩张共济失调症(ataxia telangiectasia,AT)患者皮肤的成纤维细胞系AT细胞(ATSBIVA)端粒酶活性的影响。方法:以源于正常人皮肤的成纤维细胞系GM细胞(GM0639)为对照,应用基于PCR的端粒重复扩增技术(telomeric repeat amplification protocal,TRAP)与高效液相色谱(HPLC)技术,定量分析细胞分别经0、1、3、5 Gy 60Coγ射线照射后以及经3 Gy 60Coγ射线照射后继续培养2、24、48、72 h,AT、空载体AT、ATM -AT和GM细胞的端粒酶活性的变化。结果:未照射时,除GM细胞外,AT、空载体AT、ATM -AT细胞均呈现较高的端粒酶活性表达,但ATM -AT细胞的端粒酶活性明显低于AT和空载体AT细胞的端粒酶活性(P<0．01),而后二者无明显差异(P> 0．05);电离辐射照射后,AT、空载体AT、ATM -AT和GM细胞的端粒酶活性均呈剂量依赖性和时间依赖性增强,且在相同剂量点与时间点,ATM -AT细胞的端粒酶活性高于GM细胞(P<0．01)(除5 Gy计量点外),但低于AT和空载体AT细胞(P<0．01),而后二者无明显差异(P>0．05)。结论:电离辐射可诱导细胞端粒酶活性表达;并且细胞端粒酶活性水平随剂量与时间的增加而增加;ATM可下调AT细胞端粒酶活性水平。推测端粒酶参与电离辐射诱导DNA损伤的修复。     

靶向沉默ATM基因对人肺腺癌A549细胞放射敏感性影响

目的 探讨质粒介导RNA干扰抑制ATM基因表达对人肺腺癌A₅₄₉细胞放射敏感性影响。方法 构建ATM基因小分子干扰RNA (siRNA)真核表达质粒pSilencer2.1-ATM并转染A₅₄₉细胞(阳性组),转染pSilencer2.1-nonspecific质粒为阴性组,未转染为对照组。RT-PCR和蛋白印迹法分别检测ATM基因mRNA及蛋白表达,细胞克隆形成实验观察细胞放射敏感性变化,流式细胞仪检测细胞周期和细胞凋亡。结果 成功构建了ATM基因siRNA真核表达质粒。RT-PCR和蛋白印迹法证实阳性组细胞ATM基因表达下调,阳性组、阴性组的放射增敏比(D₀值比)分别为1.50、1.01,流式细胞仪检测显示阳性组G₁、G₂+M期细胞比例减少[51.27%∶61.85%(P=0.012)、6.34%∶10.91%(P=0.008)],细胞凋亡率则高于对照组、阴性组[49.31%∶13.58%(P=0.000)、49.31%∶13.17%(P=0.000)]。结论 ATM基因沉默可增加A₅₄₉细胞的放射敏感性,其机制可能与细胞周期变化及凋亡有关。     

Twist基因真核表达载体的构建和表达检测

[目的]构建Twist基因读码框架至真核表达载体pCDNA3．1，为进一步研究Twist蛋白的功能做材料准备。[方法]采用RT-PCR方法，以正常人子宫体组织cDNA为模板，扩增Twist基因全长读码框架，并构建到真核表达载体pCDNA3．1中。[结果]经过克隆测序结果证实，成功将Twist基因读码框架插入真核表达载体pCDNA3．1的相应位置：将pCDNA-Twist载体转染细胞系后，发现Twist蛋白在细胞中得到高水平的表达。[结论]Twist真核表达载体的成功构建，为深入研究Twist蛋白对肿瘤转移的作用提供了理想的材料。     

人核糖体蛋白L6对白血病K562/A02细胞耐药性的影响

目的观察核糖体蛋白L6(RPL6)表达的改变对白血病耐药性的作用。方法通过RT-PCR方法获得RPL6cDNA序列,用真核表达载体pcDNA3.1( )分别构建正向插入和反向插入的RPL6cDNA重组质粒。以脂质体将正义RPL6cDNA真核表达质粒转染K562细胞,将反义RPL6cDNA真核表达质粒转染K562/A02细胞,以MTT观察其对化疗药物耐药性的作用。结果转染正义RPL6cDNA真核表达质粒后,K562细胞对阿霉素的耐药性比未转染的K562细胞增强3.25倍,转染反义RPL6cDNA真核表达质粒后,K562/A02细胞对阿霉素的耐药性比未转染的K562/A02细胞降低62%。结论RPL6基因过表达在K562/A02细胞耐药性的形成中起重要作用。     

共表达p53、p16基因真核表达载体的构建及其在K562细胞中的表达

[目的]构建p53、p16基因真核表达载体,观察其在K562细胞中的表达。[方法]设计并构建包含野生型p53cDNA、p16cDNA的真核双表达载体pBudCE4．1-53—16,脂质体法转染K562白血病细胞,用间接免疫细胞化学法、Western blot等方法鉴定外源性p53及p16的表达情况。[结果]构建成功p53、p16基因双表达真核载体,体外成功转染入K562细胞,检测到K562细胞外源性p53、p16蛋白的表达。[结论]重组质粒pBudCE4．1-53-16体外转染K562细胞后．目的基因能够在细胞中同时表达,为进一步研究两种基因用于肿瘤治疗奠定基础。     

人THANK基因转染人肝癌细胞及生物学特性

[目的]将人THANK基因转染入人肝癌细胞SMMU-7721中稳定表达,检测转染后细胞的生物学特性变化.[方法]将由pMD18-T-THANK质粒上获得的THANK全长cDNA克隆到真核表达载体pcDNA3.1中;用脂质体法将pcDNA3-THANK导入人肝癌细胞株SMMU-7721中,G418筛选克隆,再以RT-PCR及Western blot检测THANK的表达,流式细胞仪及细胞毒杀伤实验分析转染后细胞的生物学特性变化.[结果]THANK在7721细胞中的表达可抑制7721细胞的生长,使肿瘤细胞的生长停滞于S期,且可在体外诱导强烈的细胞毒性T淋巴细胞(CTL)反应,人外周血单个核细胞(PBMC)对THANK-7721细胞的杀伤活性明显增强.THANK的转染与IFN-γ协同可增强7721细胞的凋亡比例.[结论]THANK基因的转染改变了7721细胞的生物特性,在体外诱导PBMC的CTL反应,有可能用于肝癌的免疫治疗.     

pEGFP-C3/Smad3真核表达质粒的构建及在MCF-7乳腺癌细胞中的转染

目的构建pEGFP-C3/Smad3真核表达质粒, 并进行鉴定。为进一步研究Smad3在抑制乳腺癌生长机制中起到的作用提供实验基础。方法 利用RT-PCR方法从MCF-7乳腺癌细胞中获得cDNA, 应用PCR方法扩增、提取 Smad3的基因片段, 酶切后与pEGFP-C3真核表达载体连接, 构建pEGFP-C3/Smad3真核表达质粒, 进行测序、酶切鉴定, 表明质粒构建成功。将构建的质粒瞬时转染至MCF-7乳腺癌细胞中, 通过荧光倒置显微镜观察、RT-PCR技术及Westen blot技术鉴定转染是否成功。结果 本实验成功构建了pEGFP-C3/Smad3真核表达质粒。结论 pEGFP-C3/Smad3真核表达质粒瞬时转染到MCF-7细胞中, 可使Smad3在基因与蛋白水平的表达显著上调。     

Chronic radiation enteritis: a community hospital experience

A retrospective study was undertaken to evaluate the operative management of patients with chronic radiation enteropathy. Thirty-eight affected patients from 1974 to 1986 were reviewed. Patients with recurrent cancer responsible for symptoms were excluded. Seventy-one percent of patients presented with bowel obstruction. Twenty-one patients were treated with bowel resection, while 17 were treated with a bypass procedure or diverting ostomy alone. Overall morbidity was 45%, and postoperative mortality was 16%. Patients in the bypass group were significantly older than those in the resection group (70.3 vs. 55.5 years, P = .024), suggesting that age may have been a determinant of the procedure performed. In our study there was no difference in outcome based on preexisting vascular disease, tumor site, type of procedure performed, or radiation dose. We conclude that resection is the procedure of choice in cases of chronic radiation enteritis requiring surgery except in cases with dense adhesions when enteroenterostomal bypass is a viable alternative.     

Numbers and proportions of leukemias in young people and adults induced by radiation of natural origin

Natural sources contribute a large fraction of the radiation exposure of the general public. Under the linear no-threshold hypothesis risk decreases in proportion to decreasing dose without a threshold. We use recent estimates of doses to the red bone marrow to calculate the number and proportion of cases of leukemia in England induced by natural radiation. We calculate that about 5% of cases of leukemia, excluding chronic lymphocytic leukemia, up to the age of 80 years are induced by this background radiation. In young people up to the age of 25 years the attributable fraction is about 15%, substantially lower than a previous estimate.     

Histopathological effects of intraoperative radiotherapy on pancreas and adjacent tissues: a postmortem analysis

Intraoperative radiotherapy (IORT) has been utilized in the treatment of resectable and unresectable pancreatic carcinoma at the National Cancer Institute. Detailed autopsy analyses of the radiation effects on the pancreas and adjacent tissues were performed on 13 patients dying at various times following therapy. IORT can induce a progressive retroperitoneal fibrosis and fibrosis of the porta hepatis in patients with resectable pancreatic carcinoma. In unresectable pancreatic carcinoma, the major expression of intraoperative irradiation with external beam irradiation is a progressive fibrosis of the pancreas with vascular sclerosis, nerve degeneration, atrophy of acinar cells, and atypical changes in the ducts of the pancreas, as well as degenerative changes of the pancreatic tumor.     

Progress and controversies: Radiation therapy for prostate cancer

Answer questions and earn CME/CNE Radiation therapy remains a standard treatment option for men with localized prostate cancer. Alone or in combination with androgen‐deprivation therapy, it represents a curative treatment and has been shown to prolong survival in selected populations. In this article, the authors review recent advances in prostate radiation—treatment techniques, photon versus proton radiation, modification of treatment fractionation, and brachytherapy—all focusing on disease control and the impact on morbidity. Also discussed are refinements in the risk stratification of men with prostate cancer and how these are better for matching patients to appropriate treatment, particularly around combined androgen‐deprivation therapy. Many of these advances have cost and treatment burden implications, which have significant repercussions given the prevalence of prostate cancer. The discussion includes approaches to improve value and future directions for research. CA Cancer J Clin 2014;64:389–407. © 2014 American Cancer Society.     

依济复防治急性放射性皮肤和粘膜反应的初步临床观察

目的　观察依济复在预防及治疗放射性皮炎和粘膜炎中的作用。方法　 1999年 10月～ 2 0 0 0年 9月 ,应用依济复喷剂预防及治疗 5 0例行放射治疗患者 (预防治疗组 ) ,并设同等数量常规处理的患者为对照组。结果　预防治疗组Ⅱ度以上放射性皮炎发生率为 2 0 %,对照组 93.3%,两组有显著性差异 (P <0 .0 0 5 ) ;预防治疗组Ⅱ度以上放射性粘膜炎发生率为 10 %,对照组为 90 %,两组也具有显著性差异 (P <0 .0 0 5 )。结论　依济复对Ⅱ度以上放射性皮炎和粘膜炎的预防有一定作用 ,对放射性皮炎和粘膜炎的治疗效果显著 ,可以将放射性皮炎和粘膜炎基本控制在Ⅱ度以内 ,起到止痛、止痒、促进伤口愈合等作用。     

Fast neutron irradiation for prostate cancer

The purpose of this study was to summarize the progress made using fast neutron irradiation in the treatment of prostate cancer at Wayne State University between 1991 and the year 2001. The results of three Phase II studies and one Phase III study involving nearly 700 patients is summarized in this paper. The Phase II studies were dose finding studies looking at doses of 15, 9, 10, and 11nGy, respectively. The randomized protocol was a study of sequence looking at the results of treating patients with neutron first versus neutron radiation last. The results demonstrated that the best combination of tumor control probabilities and normal tissue complications was found in a mix of approximately 50% neutrons and 50% photons. Thus, the standard doses become 10nGy and 40Gy of photons. The randomized trial demonstrated that the sequence has significant importance and the disease-free survival was 93% for patients treated with neutrons first versus 73% for patients treated with neutrons last. There was no difference in the rate of acute or chronic complications. Finally, an analysis was performed demonstrating which patients may best benefit from the use of neutron irradiation. It was shown that patients with one, two, or three adverse risk factors had a significant improvement in disease-free survival when part of the treatment included neutron radiation versus standard photon radiation alone. Neutron radiation can be delivered safely with effort to see that it is superior to that which can be achieved by conformal photon irradiation by itself. Future work will be done to expand the role of neutron radiation in other clinical disease sites.     

体外加腔内放射治疗115例鼻咽癌的远期疗效

为探讨腔内照射在鼻咽癌治疗中的作用，作者自１９７３年３月至１９７８年８月，对１１５例鼻咽癌患者作体外加腔内放射治疗。原发灶外照射剂量５３～７６Ｇｙ／６～８周，腔内照射剂量１５～４０Ｇｙ／１～２次。全部病人均随访１０年以上，３年、５年和１０年生存率分别为７１．３０％、５７．３９％、４０．００％。影响预后的主要因素是临床分期，Ⅰ、Ⅱ、Ⅲ和Ⅳ期的５年生存率分别为８３．３３％、６７．４４％、５７．１７％和０。作者认为腔内照射对Ｔ１、Ｔ２和部分Ｔ３期患者是一种有效的治疗方法。