血管生长抑制因子angiostatin在昆虫细胞中的表达及活性研究

背景与目的:抑制新生血管的形成,有助于抑制肿瘤的生长,减少和预防肿瘤转移的发生。血管抑素(angiostatin)是一种重要的内源性新生血管生成抑制剂,对血管内皮细胞增殖有较强的抑制作用。为获得具有高活性的Angiostatin表达,本研究通过杆状病毒表达系统表达重组的angiostatin,分析其在昆虫细胞中表达和生物活性。方法:用带有angiostatin的杆状病毒转移载体pBlueBacHis2B和病毒DNA共同转染Sf9细胞,构建重组病毒,蚀斑实验筛选,PCR分析确定后扩增产生大量高滴度的病毒贮存液;用SDS-PAGE电泳和Westernblot对感染不同时间后分泌的重组蛋白做时间表达分析;用ProBondTM纯化系统对表达的重组angiostatin进行纯化,分光光度计确定蛋白含量,SDS-PAGE电泳确定蛋白纯度;采用MTT法测定重组蛋白angiostatin对原代培养的人脐静脉内皮细胞(HUVEC)的抑制作用而后通过鸡胚尿囊膜实验进一步证实其抗血管形成作用。结果:成功构建了滴度为2×108pfu/ml的angiostatin重组杆状病毒,并在昆虫细胞Sf9中高效表达了分子量为53ku的angiostatin重组蛋白,纯度约为90%,重组angiostatin蛋白不仅在体外显著抑制内皮细胞的生长(IC50为2.3μg/ml),而且显著抑制鸡胚尿囊膜血管的生长。结论:此系统可制备高滴度angiostatin重组杆状病毒贮存液,并在Sf9昆虫细胞中高效表达该重组蛋白,经在体和离体细胞学实验验证其对内皮细胞的增殖具有抑制作用。     

重组人血管抑素的克隆、表达及活性测定

目的：研究血管抑素（ａｎｇｉｏｓｔａｔｉｎ）的临床应用价值,将其开发为多肽类药物。方法：ＲＴ－ＰＣＲ从胎儿肝脏钓取人血管抑素全编码区ｃＤＮＡ,使用Ｐｉｃｈｉａ分泌型酵母表达系统表达重组人血管抑素,重组蛋白经肝素亲和层析纯化后,以鸡胚尿囊膜法血管生成实验及小鼠创伤愈合实验检测活性。结果：重组人血管抑素在酵母系统中得到较高量表达（５ｍｇ／Ｌ）,经肝素亲和层析纯化,ＳＤＳ－ＰＡＧＥ显示分子量为４３ｋＤ。活性实验表明,重组蛋白能够抑制新生管形成、抑制伤口愈合。结论：重组人血管抑素在酵母系统中实现高效表达,并具有很好的生物学活性。     

血管内皮抑素的原核表达及多克隆抗体的制备

背景与目的：本实验拟在原核系统中表达并纯化人血管内皮抑素（endostatin),制备鼠抗人血管内皮抑素多克隆抗体。方法：设计引物扩增内皮抑素的cDNA,重组人原核表达载体并在大肠杆菌中诱导表达,应用纯化产物免疫3只小鼠。结果：构建成制备人血管内皮抑素的原核表达载体pQE-30,并转化入大肠杆菌BL21中得到表达产物,经Ni亲和层析柱纯化后进行SDS-PAGE鉴定结果为单一组分。利用纯化的人血管内皮抑素成功制备高滴度的鼠抗人血管内皮抑素多克隆抗体,并由Western blot分析证实。结论：高纯度表达产物及多克隆抗体的制得为今后的研究提供了素材。     

血管生成抑素的原核表达及多克隆抗体的制备

目的:在原核系统中表达并纯化人血管生成抑素（angiostatin）,制备鼠抗人血管生成抑素多克隆抗体。方法:设计引物扩增angiostatin的cDNA,然后亚克隆入原核表达载体pQE,并转化入大肠杆菌BL21中诱导表达并纯化,再用纯化的人血管生成抑素免疫小鼠并制备多抗。结果:通过重组质粒酶切和测序分析等方法,筛选出重组阳性克隆,转化入大肠杆菌BL21中诱导表达并纯化成功,再利用纯化的人血管生成抑素制备成功鼠抗人血管生成抑素多克隆抗体。结论:表达产物及多克隆抗体为下阶段深入研究提供了重要的实验材料。     

重组IFN-α2a与HSV-tk/GCV系统协同作用增强对于卵巢癌细胞系SKOV3杀伤作用的体外研究

目的：在原核系统中表达并纯化人血管生成抑素（angiostatin),制备鼠抗人血管生成抑素多克隆抗体。方法：设计引物扩增angiostatin的cDNA,然后亚克隆入原核表达载体pQE,并转化入大肠杆菌BL21中诱导表达并纯化,再用纯化的人血管生成抑素免疫小鼠并制备多抗。结果：通过重组质粒酶切和测序分析等方法,筛选出重组阳性克隆,转化入大肠杆菌BL21中诱导表在并纯化成功,再利用纯化的人血管生成抑素制备成功鼠抗人血管生成抑素多克隆抗体。结论：表达产物及多克隆抗体为下阶段深入研究提供了重要的实验材料。     

重组人内皮抑素的纯化及抗肿瘤活性研究

[目的]从高效表达的基因工程菌中纯化重组人内皮抑素,对其抗肿瘤活性进行研究.[方法]经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组人内皮抑素在大肠杆菌基因工程菌中以包涵体形式高效表达.通过凝胶层析纯化重组内皮抑素蛋白.应用鸡胚绒毛尿囊膜实验(CAM),肺癌细胞MTT试验及细胞迁移抑制实验,裸鼠皮下移植喉癌抑瘤实验、病理组织切片、免疫组化指标的测定等检测研究重组人内皮抑素的抗肿瘤活性.[结果]重组内皮抑素的复性率可达40%.重组内皮抑素在体外直接抑制肺癌细胞的增殖及迁移.用药21天,对裸鼠皮下移植喉癌的抑瘤率达到40.66%,HE染色、免疫组化及CAM实验表明其对肿瘤组织新生血管的生成有较强的抑制作用.[结论]重组人内皮抑素具有抑制肿瘤组织新生血管生成和直接抑制肿瘤细胞生长和迁移的双重抗肿瘤活性.     

血管形成抑制因子HIAF-1的克隆及其抗肿瘤作用

目的　克隆和表达一种新的血管形成抑制因子 ,并研究其抗肿瘤活性。方法　应用逆转录聚合酶链反应从人胎儿肝脏组织中克隆到一种新的血管形成抑制因子 (humaninhibitingangiogenesisfactor 1,HIAF 1) ,经测序后克隆到表达载体pET30a( )上 ,在E .coliBL2 1∶DE3菌中表达。用MTT法体外检测HIAF 1对内皮细胞的作用 ,采用小鼠乳癌自发性肺转移模型IVTA2MA 891,体内研究重组蛋白对肿瘤血管形成的作用和抑瘤作用。结果　从胎儿肝脏组织中首次克隆到人源血管形成抑制因子HIAF 1,经测序为人胶原ⅩⅧC 末端的一部分。在E .coli细胞中高效表达了该因子的重组蛋白 ,蛋白表达量为 88mg/L。体外实验表明HIAF 1能抑制内皮细胞的增殖 ,IC50 值为 7.5 μg/ml。动物实验表明该重组蛋白能够显著抑制肿瘤血管形成 ,原发瘤的生长 (抑瘤率为 46 .6 % )及肿瘤的转移 (抑制转移率为 6 8.9% )。结论　成功的克隆了血管形成抑制因子HIAF 1,其能够抑制肿瘤血管的形成 ,在肿瘤的治疗中显示出良好的应用前景。     

重组人canstatin表达产物的抗肿瘤活性鉴定

 目的　正确构建人canstatin原核表达载体 ,诱导表达重组人canstatin融合蛋白 ,并对其活性鉴定。方法　将人canstatincDNA亚克隆入 pQE30 ,构建重组质粒 pQE30 canstatin ;转化M 15 [pREP4] 中 ,IPTG诱导表达 ,纯化回收表达产物 ,复性后用Lewis肺癌移植瘤模型对其活性鉴定。结果　成功构建人canstatincDNA表达载体 ,纯化回收 ,获得高纯度canstatin重组蛋白。纯化产物在体内能抑制Lewis肺癌移植瘤血管的生成 ,从而抑制肿瘤生长和转移 ,抑瘤率 (% )为 6 0 .0 % ,转移抑制率 (% )为 74 .3%。结论　 (1)成功构建了原核表达载体 pQE30 canstatin。 (2 )重组人canstatin融合蛋白在原核表达系统中高水平表达 ,并获得高纯度canstatin重组蛋白。 (3)重组canstatin融合蛋白可抑制Lewis肺癌移植瘤血管的生成 ,从而抑制肿瘤生长和转移活性。     

血管发生抑制因子 restin的克隆表达及其抗血管活性的初步研究

目的：克隆人血管发生抑制因子restin(hRs)，在E.coli中融合表达，并测定其抗血管活性。方法：用RT-PCR法从中国人胎盘组织中扩增hRs基因，重组人pGEM-T载体中并测定鉴定，构建原核表达载体pGEX-hRS，表达融合蛋白GST-hRS。融合蛋白经亲和纯化及凝血酶切后，采用鸡胚绒毛膜尿囊膜试验检测其抗血管生成活性。结果：RT-PCR产物为564bp，测序结果与Genbank中胶原XV(COL15A1)的C端序列一致，但在21位（TCT→TCG）引起丝氨酸的同义突变，82位（ACA→TCA）引起丝氨酸突变为苏氨酸。诱导表达的人GST-hRS融合蛋白经凝血酶切后，分子量为20kD，具有抗血管生成活性。结论：hRS的成功克隆、表达为抗血管生成治疗实体瘤的研究奠定了实验基础 。     

重组人血管内皮抑素与多西紫杉醇不同顺序用药调控移植瘤组织MMP及抗瘤效应观察

背景与目的 通过药物干预性动物实验,观察重组人血管内皮抑素(rh-endostatin)与多西紫杉醇不同顺序联合用药后MMP-2及相关因子的变化及其对移植瘤生长与血管生成的影响,探索两药联合的作用机制和最佳抗肿瘤方案.方法 建立肺腺癌A549荷瘤裸鼠模型,分两个阶段进行实验.第一阶段:将荷瘤小鼠随机分为重组人血管内皮抑素组(重组人血管内皮抑素400μg·d-1,d1-d14)和多西紫杉醇组(多西紫杉醇10 mg·kg1·3d-1,d1-d14);第二阶段:将荷瘤小鼠随机分为同时用药组(重组人血管内皮抑素400μg·d-1、d1-d35,多西紫杉醇10mg·kg-1·3d-1、d1-d19)、先重组人血管内皮抑素组(重组人血管内皮抑素400μg·d-1、d1-d35,多西紫杉醇10 mg·kg-1·3d-1、d16-d34)和模型组.实验中动态测量移植瘤体积,结束后以免疫组化方法检测MMP-2、TIMP-2、EMMPRIN表达并计数微血管密度(MVD).结果 两单药组比较,重组人血管内皮抑素组MMP-2和EMMPRIN表达下调较多西紫杉醇组(P=0.024,P=0.081)明显,两组间TIMP-2表达无明显差异(P＞0.05).联合组用药结束时,同时用药组与先重组人血管内皮抑素组的移植瘤体积小于模型组(P＜0.001,P=0.003),且MMP-2表达均明显下调、微血管数减少(P＜0.05),但同时用药组对肿瘤生长的抑制较先重组人血管内皮抑素组明显;与模型组相比,同时用药组TIMP-2上调(P=0.001)、EMMPRIN下调(P=0.018),先重组人血管内皮抑素组未见相似结果.结论 同时用药方案可以从TIMP-2、EMMPRIN两个环节下调MMP-2的表达,从而更好地抑制肿瘤生长.     

New and emerging radiosensitizers and radioprotectors

The combination of chemotherapy and radiation has led to clinical breakthroughs in several disease sites, and current work continues to define optimum combinations of proven chemotherapy as well as more recently available, noncytotoxic agents. Administration of systemic therapies allows modulation of radiation response to improve tumor control (radiosensitization) or to prevent normal tissue toxicity (radioprotection). Substantial progress has been made in identifying the targets of standard chemotherapeutic radiation sensitizers and protectors as well as in the introduction of a new generation of molecularly targeted therapies in combination with radiation. We have reviewed the most recent, predominantly early phase clinical trials combining systemic agents with radiation. Although the proof of an improved schedule ultimately needs to come from well-run Phase III trials, the search among schedules could be shortened by the use of surrogate endpoints such as presence of active drug metabolites in the tumor. This has been accomplished only in a few cases and needs to become a more standard part of radiation sensitizer and protector trials.     

Religiosity and Physical and Emotional Functioning Among African American and White Colorectal and Lung Cancer Patients

The literature suggests that religiosity helps cope with illness. The present study examined the role of religiosity in functioning among African Americans and Whites with a cancer diagnosis. Patients were recruited from an existing study and mailed a religiosity survey. Participants (N = 269; 36% African American, 56% women) completed the mail survey, and interview data from the larger cohort was utilized in the analysis. Multivariate analyses indicated that in the overall sample religious behaviors were marginally and positively associated with mental health and negatively with depressive symptoms. Among women, religious behaviors were positively associated with mental health and negatively with depressive symptoms. Religiosity was not a predictor of study outcomes for men. Among African Americans, religious behaviors were positively associated with mental health and vitality. Among Whites, religious behaviors were negatively associated with depressive symptoms. These findings suggest a mixed role of religious involvement in cancer outcomes. The current findings may have applied potential in the areas of emotional functioning and depression.     

Fruit and vegetables and cancer risk

The possibility that fruit and vegetables may help to reduce the risk of cancer has been studied for over 30 years, but no protective effects have been firmly established. For cancers of the upper gastrointestinal tract, epidemiological studies have generally observed that people with a relatively high intake of fruit and vegetables have a moderately reduced risk, but these observations must be interpreted cautiously because of potential confounding by smoking and alcohol. For lung cancer, recent large prospective analyses with detailed adjustment for smoking have not shown a convincing association between fruit and vegetable intake and reduced risk. For other common cancers, including colorectal, breast and prostate cancer, epidemiological studies suggest little or no association between total fruit and vegetable consumption and risk. It is still possible that there are benefits to be identified: there could be benefits in populations with low average intakes of fruit and vegetables, such that those eating moderate amounts have a lower cancer risk than those eating very low amounts, and there could also be effects of particular nutrients in certain fruits and vegetables, as fruit and vegetables have very varied composition. Nutritional principles indicate that healthy diets should include at least moderate amounts of fruit and vegetables, but the available data suggest that general increases in fruit and vegetable intake would not have much effect on cancer rates, at least in well-nourished populations. Current advice in relation to diet and cancer should include the recommendation to consume adequate amounts of fruit and vegetables, but should put most emphasis on the well-established adverse effects of obesity and high alcohol intakes.     

VEGF及KDR在大肠腺瘤和腺癌中的表达及意义

目的：探讨VEGF和KDR在大肠腺瘤和大肠腺癌中的表达及临床病理特征的关系。方法：大肠腺瘤和大肠腺癌组织标本各100例,采用免疫组织化学染色法检测VEGF和KDR在标本中的表达情况。结果：VEGF和KDR在大肠腺癌组中的阳性表达明显高于大肠腺瘤组（P〈0.05）;在正常大肠黏膜均未见VEGF和KDR表达的阳性染色;VEGF阳性表达组中KDR的阳性表达率为70%,显著高于VEGF阴性表达组中KDR的阳性表达率16%,两组比较有统计学意义（P〈0.01）。结论：大肠腺癌组织中KDR的表达与肿瘤大小、转移情况、浸润深度密切相关;VEGF和KDR在大肠腺瘤中的表达与患者的年龄、性别及分型均无相关性,而与增生程度相关（P〈0.05）。在大肠腺癌患者中VEGF及KDR表达更高,二者具有协同效应。     

肾上腺素能β受体与肿瘤生长及转移

大量研究表明肿瘤细胞可表达β受体，而一些神经递质、药物和社会心理因素可能通过β受体影响肿瘤的生长和转移，β受体激动剂、β受体阻滞剂以及抑郁等社会心理因素可加强或削弱这种作用。这为表达β受体肿瘤的治疗开辟了新的道路，提供了新的治疗靶点。     

Occupational and environmental radiation and cancer

Epidemiologic evidence on the relation between occupational and environmental radiation and cancer is reviewed. Studies of pioneering radiation workers, underground miners, and radium dial painters revealed excess cancer deaths and contributed to the setting of radiation protection standards and to theories of carcinogenesis. Occupational exposures today are generally much lower than in the past, thus any associated increases in cancer will be difficult to detect. Pooling investigations of these more recently exposed workers, however, has the potential to validate current estimates of risk used in radiation protection. New information on the effects of chronic radiation exposure also may come from studies in the former Soviet Union of Chernobyl clean-up workers and of workers at the Mayak nuclear facilities. Studies of environmental radiation exposures, other than radon, are largely inconclusive, due mainly to the difficulties in detecting the low risks associated with low dose exposures. Thyroid cancer, however, has been linked to environmental radiation from the Chernobyl accident and from nuclear weapons tests. Low-level radiation released during normal operations at nuclear plants has not been found to increase cancer rates in surrounding populations. Radon, a human carcinogen, is the most ubiquitous exposure to human populations; remediating high residential-radon levels is recommended, recognizing that the exposure can never be removed completely because it occurs naturally.     

Cell and tissue interactions in carcinogenesis and metastasis and their clinical significance

This review describes a new vision for future directions in the study of metastatic cancer biology and pathology. It is based upon clinical and experimental observations on the constituent cell lineages within a neoplasm and on tumour-host interactions. The vision incorporates information from studies in population biology, developmental biology and experimental pathology as well as investigations upon human malignant disease. The assembled information reveals that invasion and metastasis are supra-cellular manifestations of "emergent behavior" among combinations of normal and malignant cell lineages in vivo. Emergent behavior is a combinatorial interactive process in which a population displays new traits which cannot be achieved by individuals acting separately and which subside when the specific population mix disaggregates. Disruption of such pathological interactions in the field of a developing primary or secondary tumour is, therefore, required to disable the malignant population and arrest progression without tissue destruction. These conclusions originate, in part, from principles which govern the sociobiology and group behavior of bees, ants, fish, birds and human societies. In all these social organisms, external factors can disrupt signaling mechanisms and induce expanding self-perpetuating rogue behavior, leading to social disintegration. These principles also apply to cellular societies composing higher animals, which likewise need intrinsic rules to maintain social order and avoid anarchy, and recognition of this is essential for advancing future research on the mechanisms involved in carcinogenesis and metastasis. Summarised evidence is presented here to support the conclusion that miscommunications between cells and tissues in the region of the developing tumour and its metastases are the main direct perpetrators of malignant disease. Genetic lesions (mutations, deletions, translocations, reduplications, etc.), commonly seen in cancers, can significantly disrupt important molecular pathways in the networks of communications needed to sustain orderly tissue/organ structure and function. However, genetic lesions can also, themselves, be induced by abnormal cell interactions initiated by extrinsic carcinogenic agents such as chemicals, viruses, hormones and radiation. The evidence shows that, irrespective of the initiating cause, it is this miscommunication in the region of a developing tumour and its metastases that is ultimately responsible for the emergence and progression of the disease. The article describes how this information collectively, provides a framework for designing specific novel therapeutic approaches targeting the cell and tissue interactions driving tumour metastasis and its manifold effects on the whole body.     

Vitamin D and melanoma and non-melanoma skin cancer risk and prognosis: A comprehensive review and meta-analysis

Vitamin D is formed mainly in the skin upon exposure to sunlight and can as well be taken orally with food or through supplements. While sun exposure is a known risk factor for skin cancer development, vitamin D exerts anti-proliferative and pro-apoptotic effects on melanocytes and keratinocytes in vitro. To clarify the role of vitamin D in skin carcinogenesis, we performed a review of the literature and meta-analysis to evaluate the association of vitamin D serum levels and dietary intake with cutaneous melanoma (CM) and non-melanoma skin cancer (NMSC) risk and melanoma prognostic factors. Twenty papers were included for an overall 1420 CM and 2317 NMSC. The summary relative risks (SRRs) from random effects models for the association of highest versus lowest vitamin D serum levels was 1.46 (95% confidence interval (CI) 0.60–3.53) and 1.64 (95% CI 1.02–2.65) for CM and NMSC, respectively. The SRR for the highest versus lowest quintile of vitamin D intake was 0.86 (95% CI 0.63–1.13) for CM and 1.03 (95% CI 0.95–1.13) for NMSC. Data were suggestive of an inverse association between vitamin D blood levels and CM thickness at diagnosis. Further research is needed to investigate the effect of vitamin D on skin cancer risk in populations with different exposure to sunlight and dietary habits, and to evaluate whether vitamin D supplementation is effective in improving CM survival.