125I-UdR提高内皮抑素基因的抑瘤作用

目的：探讨125I-UdR提高内皮抑素基因对大鼠种植癌模型的抑制效应。方法：制作肿瘤株Walker-256细胞大鼠皮下种植癌的动物模型并分为1-4组（对照组、125I-UdR组、Endostatin组和Endostatin＋125I-UdR组）,每组20只,通过实体瘤内注射,分别给与相同体积生理盐水125I-UdR、内皮抑素基因及125I-UdR和内皮抑素基因混合物,测量肿瘤治疗前体积（V0）和治疗后不同时间体积（Vt）,计算10d、20d的肿瘤生长率（f=Vt／V0）,观察各组肿瘤在光镜下的变化。结果：各组大鼠10d、20d肿瘤生长率为：（11．03±1．08、27．35±1．08）,（4．02±0．79、7．58±2．98）,（3．88±0．26、7．02±2．75）,（2．72±1．01、2．94±1．26）,2、3、4组的肿瘤生长率明显小于l组（P〈0．001）;2、3组之间肿瘤生长率差别不明显（P〉0．05）,4组肿瘤生长率小于2、3组（P〈0．01）.结论：通过实体瘤内注射的方法给与125I-UdR、内皮抑素基因及两者混合物后,能够明显抑制肿瘤的生长,内皮抑素基因和125I-UdR联合治疗在抑制肿瘤生长方面作用更加显著。     

重组人内皮抑素联合TP方案对MCF-7裸鼠移植瘤的影响

目的:探讨重组人内皮抑素联合TP方案对MCF-7裸鼠移植瘤模型血管生成及肿瘤生长的作用.方法:将36只荷瘤鼠随机分为A、B两大组,A、B两大组内均设有单一化疗组、联合用药组及对照组;单一化疗组给予紫杉醇 顺铂(TP方案).联合用药组给予重组人内皮抑素 TP,对照组等体积生理盐水;比较A大组内各组裸鼠的抑瘤率,肿瘤生长曲线.ELISA方法检测血清血管内皮生长因子(VEGF),取肿瘤组织病理切片观察微血管密度(MVD)、凋亡指数,并观察B组所有动物生存期.结果:联合用药组VEGF,MVD,较单一化疗组及对照组低,凋亡指数比单一化疗组高,有统计学差异(P＜0.05),联合用药组与化疗组生存时间的差异无统计学意义(P＞0.05),与对照组比较有统计学差异,生存时间较对照组长(P＜0.05).结论:重组人内皮抑素联合TP方案对MCF-7裸鼠移植瘤对肿瘤生长及肿瘤血管抑制优于单一化疗,但1个周期联合化疗用药不能延长生存期.     

放射治疗联合血管内皮抑制素对大鼠移植性肝癌的治疗作用

目的:探讨放射治疗联合血管内皮抑制素对大鼠移植性肝癌的治疗作用。方法:SD大鼠随机分为5组:假手术组,模型组,血管内皮抑素组,放疗组,联合组,血管内皮抑素组和联合组每日尾静脉注射浓度为10%重组人血管内皮抑素0.1 ml(20μg),共12天,联合组在第5天～10天时给予放射治疗,1次/2天。各组动物于移植术后12d处死,分离肿瘤组织备用。结果:血管内皮抑素组、放疗组、联合组均能显著抑制肿瘤生长,抑制VEGF表达,其中联合组抑制效果更为明显。结论:放射治疗联合血管内皮抑制素有明显肿瘤抑制作用,其机制可能与下调VEGF的表达有关。     

重组人内皮抑素的结构改造及抗肿瘤活性变化

内皮抑素是一种内源性的血管生成和肿瘤生长抑制剂 .运用定点突变技术对人内皮抑素基因工程菌中的内皮抑素基因进行改造 ,将内皮抑素中的GRIRGAD改为RGDRGD序列 ,提高了内皮抑素的抗肿瘤活性 .裸鼠体内抑瘤试验发现 ,野生与诱变内皮抑素的抑瘤率分别为 4 0 6 6 %和5 1 0 5 % ,诱变内皮抑素抑瘤率比野生内皮抑素提高了近 11个百分点 .病理切片HE染色诱变组肿瘤的血管生成比野生组明显减少 ,坏死灶增多 .免疫组化试验中 ,诱变组在微血管密度MVD(P <0 0 5 )、血管内皮生长因子VEGF(P <0 0 5 )、增殖细胞核抗原PCNA(P >0 0 5 )三个指标上均比野生组减小 .体外细胞实验中 ,MTT结果表明 ,野生组内皮抑素半数抑制浓度IC50 =185 μg ml,诱变组内皮抑素IC50 =2 7μg ml,诱变组抑瘤活性是野生组的 6倍 .细胞迁移抑制实验表明 ,野生组与诱变组内皮抑素均对肿瘤细胞迁移有抑制作用 ,野生组迁移抑制率为 6 8 95 % ;诱变组迁移抑制率为89 94 % .上述结果说明 ,内皮抑素除抑制血管生成外 ,对肿瘤细胞的生长和迁移也有一定的抑制作用 .诱变内皮抑素通过RGDRGD序列与整合素结合而提高了抗肿瘤活性 .     

食管鳞癌患者血清血管内皮生长因子与内皮抑素表达及其与临床病理特征的关系

目的:探讨食管鳞癌患者血清中血管内皮生长因子(VEGF)和内皮抑素(Endostatin)的表达及其与食管鳞癌临床病理特征和预后的关系。方法:采用ELISA法检测126例食管鳞癌患者和14例正常健康人血清VEGF及Endostatin表达水平。结果:126例食管鳞癌患者血清中VEGF(20.68±3.09)ug/L水平和Endostatin水平(4.96±1.72)ug/mL均显著高于正常健康人(3.82±6.28)μg/L和(1.60±0.37)μg/L(P<0.05),V/E比值也非常显著高于正常人。食管鳞癌患者血清中VEGF、endostatin水平以及V/E比值与其分化程度、P-TNM分期、病变长度、淋巴结转移状态等显著相关(P<0.01),与其年龄、性别、肿瘤部位、浸润深度等无明显关系(P>0.05)。食管鳞癌患者血清中VEGF与Endostatin表达呈非常显著正相关(r=0.594,P<0.01)。结论:食管鳞癌患者血清中VEGF、Endostatin水平升高,与食管鳞癌的恶性程度及肿瘤负荷密切相关,其两者的比值(V/E)对食管鳞癌患者预后、生物学行为评估具有重要意义。     

单基因和双基因联合治疗对小鼠B16-F10黑色素瘤生长抑制作用研究

目的研究阳离子脂质体介导mGM-csf和mFlt-kdr3基因治疗以及2个基因联合治疗对小鼠B16-F10黑色素瘤肺转移以及实体瘤的生长抑制作用。方法通过尾静脉注射法和皮下注射法分别将10~5个以及10~6个对数生长期黑色素瘤细胞注入BALB/c小鼠体内,构建小鼠肺转移模型和腋下实体瘤模型。将2组模型小鼠分别分成5组:mFlt-kdr3治疗组、mGM-csf治疗组、联合治疗组(1次mFlt-kdr3,2次mGM-csf)、H1299脂质体质粒对照组和生理盐水对照组。肺转移模型小鼠建模3周后,尾静脉给药治疗,每次给药80μL,每次间隔1 d,共3次。完成后3 d,解剖取出小鼠肺组织并对肿瘤灶进行计数、苏木精-伊红染色。实体瘤模型小鼠在建模1周后开始瘤体穿刺给药,每次给药25μL,每次间隔1 d,共9次,每次给药前用游标卡尺测量瘤体的长、短径。结果肺转移治疗实验中,治疗组平均肿瘤个数显著少于对照组(P<0.05),且联合治疗组肿瘤个数最少,并且肺部结构完整,基本未见明显的肿瘤灶,可以看到明显的肺泡结构。实体瘤模型实验中,治疗组瘤体平均体积显著小于对照组(P<0.05),且在观察的时间内联合治疗组瘤体平均体积呈现明显的下降趋势,治疗组的疗效价值为联合治疗组>mFlt-kdr3治疗组>mGM-csf治疗组。结论GM-csf和Flt-kdr3基因药物对黑色素瘤具有协同治疗效用,可以明显抑制肺部肿瘤灶的形成和实体瘤的生长。     

原发性口腔鳞癌组织及血清中内皮抑素表达的意义

目的:探讨原发性口腔鳞癌患者组织和血清中内皮抑素表达及与肿瘤分期、分级的关系。方法:采用免疫组化方法检测36例口腔鳞癌和12例正常口腔粘膜组织中内皮抑素表达情况。ELISA法检测36例口腔鳞癌患者术前血清内皮抑素水平,14例健康者血清做对照。结果:内皮抑素主要见于肿瘤组织细胞质。正常口腔粘膜中内皮抑素表达率为7.15%,口腔鳞癌组织中内皮抑素阳性率为76.44%,其中G1、G2、G3级阳性率分别为47.21%、79.17%、90.90%,病理分级间比较差异有统计学意义(P<0.05)。口腔鳞癌患者血清中内皮抑素水平(49.62±1.72)ng/mL显著高于健康对照者(5.60±0.37)ng/mL(P<0.05),TNM分期III、IV期肿瘤患者血清内皮抑素水平显著高于I和II期(P<0.05)。结论:口腔鳞癌患者组织和血清中内皮抑素表达显著升高,并与肿瘤分期、分级相关,检测内皮抑素表达有助于判断口腔鳞癌恶性程度。     

内皮抑素30肽基因真核表达载体的构建及实验研究

目的构建改构内皮抑素抗肿瘤相关肽（30肽）的真核表达载体pVAX1,检测该重组载体的生物学活性。方法在30肽基因的5′端加入胶原蛋白ⅩⅧ信号肽编码序列,通过PCR扩增获得目的基因30肽,并连接到质粒pVAX1中,构建表达分泌型内皮抑素的重组质粒pVAX1-30E,然后将重组质粒pVAX1-30E直接注入小鼠肿瘤组织。通过ELISA小鼠体内抑瘤实验检测目的基因的表达及其活性。结果ELISA实验表明构建的分泌型内皮抑素重组质粒pVAX1-30E能在肿瘤细胞中表达30肽,免疫组化结果表明瘤组织中表达的30肽能抑制肿瘤微血管的新生,而体内抑瘤实验表明在肿瘤部位直接注射重组质粒能抑制肿瘤生长,抑瘤率为28.19%。结论通过向瘤组织中直接注射分泌型内皮抑素重组质粒pVAX1-30E可以抑制小鼠体内肿瘤微血管新生和肿瘤生长而实现其抗肿瘤活性。     

内皮抑素可抑制血管形成和肿瘤生长

内皮抑素可抑制血管形成和肿瘤生长近年的一系列研究表明,血管形成是实体瘤生长与转移的基础。为了促进血管形成,肿瘤细胞可增加一系列血管形成因子的产生。但许多恶性肿瘤也可产生血管形成抑制因子,如血管抑素（ａｎｇｉｏｓｔａｔｉｎ）、血栓海绵素等。血管形成因子...     

重组人血管内皮抑素联合同步放化疗治疗晚期非小细胞肺癌临床研究

目的:探讨重组人血管内皮抑素联合同步化疗治疗晚期非小细胞肺癌(NSCLC)的临床疗效.方法:将我院收治的40例NSCLC患者随机分为治疗组和对照组,对照组患者采用紫杉醇化疗方案联合同步放射治疗,治疗组患者给予重组人血管内皮抑素联合同步放化疗进行治疗,治疗2个周期后对患者的近期疗效、毒副反应以及无肿瘤进展时间(TTP)等进行比较.结果:治疗组CR+PR的患者为15例,明显高于对照组的5例(P＜0.05);治疗组患者TTP为154±11d,中位生存时间为274d,而对照组TTP为108±8d,中位生存时间为179d,两组患者比较存在明显的差异性(P＜0.05);治疗组患者QOL评分明显优于对照组(P＜0.05),而两组患者毒副反应无明显的差异性(P＞0.05).结论:对晚期NSCLC患者采用重组人血管内皮抑素联合同步化疗进行治疗,可提高患者生存时间,且不会增加患者毒副反应的发生.     

Anti-tumor effect of 125I-UdR in combination with Egr-1 promoter-based IFNγ gene therapy in vivo

Although ¹²⁵I-UdR treatment of malignant tumors in animal models and patients has achieved a certain effect, the short half-life of ¹²⁵I-UdR in vivo and its cellular uptake only in S phase of the cell cycle are limiting factors with regard to tumor eradication, and therefore its combination with other applications is a promising strategy in cancer therapy. In this study, we show that ¹²⁵I-UdR radionuclide therapy in combination with Egr-1 promoter-based IFNγ gene therapy is more effective than ¹²⁵I-UdR therapy alone in suppressing tumor growth and extending survival duration in mice bearing H22 hepatomas. Combined therapy could significantly inhibit cell proliferation and tumor angiogenesis, induce apoptosis and enhance cytotoxic activities of splenic CTL of the mice. Our results suggest that ¹²⁵I-UdR in combination with Egr-1 promoter-based IFNγ gene therapy may provide novel approaches for cancer treatment.     

Evaluation of radio- and chemotoxic effects of125I-UdR on tumour growth and host survival

Summary The toxicity of 5-iodo-2-deoxyuridine (I-UdR) was assayed in male C57 BL/6J mice bearing the syngeneic mammary adenocarcinoma EO 771 by injecting different doses of cold I-UdR or 125-iodine labelled I-UdR. Host survival, tumour growth, DNA-precursor incorporation, whole-body retention and tumour activity loss rates were chosen as biological end points.There was no measurable effect on host survival up to doses of 5 µg I-UdR or 50 µCi¹²⁵I-UdR per mouse during a mean life-span of 25 days. Adjusted to a constant amount of 0.55 µg I-UdR/mouse, radiotoxicity of¹²⁵I-UdR on tumour growth (up to 17 days after implantation), tracer incorporation, whole-body and tumour retention (up to 12 days after¹²⁵I-UdR injection) could be excluded up to a dosage of 50 µCi¹²⁵I-UdR/mouse.It is concluded that in situ evaluation of tumour activity loss rates in carcinoma EO 771 is not disturbed by toxic effects of I-UdR or¹²⁵I-UdR within the dose limits mentioned.     

CELL LOSS FROM THREE ESTABLISHED LINES OF THE C3H MOUSE MAMMARY TUMOR: A COMPARISON OF THE 125I-UdR AND THE 3H-TdR-AUTORADIOGRAPHIC METHODS

The¹²⁵I-UdR method for measuring cell loss from solid tumors has been re-evaluated. The rate of tumor cell loss from three established lines (S102F, S102S and Slow) of the C3H mouse mammary tumor was determined by the ¹²⁵I-UdRmethod and the results were compared to the estimates for cell loss as determined by the combined approach of cellular ³H-TdR autoradiography and volumetric growth-rate determinations. This detailed comparison shows that the two methods complement each other but cannot substitute for one another because they give different quantitative information. The combined approach measures the flow of viable cells, as determined morphologically, from the proliferating compartment to the quiescent compartment, the quiescent compartment out of the tumor, etc., but does not evaluate the flow of degenerate cells or acellular (necrotic) debris. In contrast, the ¹²⁵I-UdR method indicates the net flow of intact cells and/or dead cells as well as debris from the tumor as the ^I25l-labeled material is lost from the tumor, but gives limited internal information. Thus, depending on the specific experiment, an investigator could choose one or the other of the methods to answer the question. Perhaps both would be desirable at times; however, in most cases, one could not substitute one method for the other. The data from the Slow tumors also indicate that in certain tumors, the quantitative information from the ¹²⁵I-UdR method may be quite limited, i.e. the confidence limits within an experiment as well as the replication error between experiments may be high.     

肌肉内转内抑素基因对肿瘤生长的抑制作用

 为研究骨骼肌及肿瘤内介导的内抑素基因转移对肿瘤生长的作用 ,利用基因克隆技术构建了内抑素基因真核表达质粒 ,应用电脉冲转移法将质粒转入转肿瘤小鼠骨骼肌或肿瘤中 .结果表明 ,内抑素基因可在骨骼肌或肿瘤内表达 ,并显著抑制肿瘤生长 .这为内抑素基因在肿瘤治疗中的应用进行了探索     

Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125

Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms.     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models

BACKGROUND: Some anaerobic and facultatively anaerobic bacteria have been used experimentally as anticancer agents because of their selective growth in tumors. In this study, we exploited attenuated Salmonella choleraesuis as a tumoricidal agent and a vector to deliver the endostatin gene for tumor-targeted gene therapy. METHODS: Attenuated S. choleraesuis carrying a eukaryotic expression plasmid encoding reporter gene was used to evaluate its abilities of tumor targeting and gene delivery in three syngeneic murine tumor models. Furthermore, S. choleraesuis carrying the endostatin expression vector was administered intraperitoneally into tumor-bearing mice, and its antitumor effect was evaluated. RESULTS: Systemically administered S. choleraesuis preferentially accumulated within tumors for at least 10 days, forming tumor-to-normal tissue ratios exceeding 1000-10,000 : 1. Transgene expression via S. choleraesuis-mediated gene transfer also persisted for at least 10 days. Host immune responses and tumor hypoxia may influence tumor-targeting potential of S. choleraesuis. When systemically administered into mice bearing melanomas or bladder tumors, S. choleraesuis carrying the endostatin expression vector significantly inhibited tumor growth by 40-70% and prolonged survival of the mice. Furthermore, immunohistochemical studies in the tumors revealed decreased intratumoral microvessel density, reduced expression of vascular endothelial growth factor (VEGF), and increased infiltration of CD8(+) T cells. CONCLUSIONS: These results suggest that tumor-targeted gene therapy using S. choleraesuis carrying the endostatin expression vector, which exerts tumoricidal and antiangiogenic activities, represents a promising strategy for the treatment of solid tumors.     

内皮抑素与肿瘤治疗

内皮抑素(endostatin)是近年来发现的,天然产生的新血管生成抑制因子。它在体内由胶原ⅩⅧ经酶解产生,可以抑制新血管生成,在肿瘤动物模型中显示出显著的抑瘤活动。通过抑制肿瘤相关的新血管的形成来治疗肿瘤,是目前出现的新疗法,内皮抑素是这种疗法中很有前景的侯选药物之一。在美国已经开始了用内皮抑素治疗肿瘤的Ⅰ期临床试验,关于它的临床前基础实验也在广泛开展。本文综述了内皮抑素在肿瘤治疗中的应用基础研究 。     

Cell loss from viable and necrotic tumour regions measured by 125I-UdR

Loss of cells from vital and necrotic areas of the syngeneic mammary adenocarcinoma EO 771 in male C57 BL/6J mice may be measured by use of 125I-labelled 5-iodo-2'-deoxyuridine (125I-UdR). Later than 50 hr after an intraperitoneal injection of 20 muCi 125I-UdR the incorporated activity of the entire tumour was externally measured and found to decrease with time after injection. The injected amount was neither chemo- nor radiotoxic. By injecting the vital dye 'light green', unstained necrotic and stained viable regions were separately excised and measured for loss of activity throughout the natural development of the labelled tumour. With the appearance of necrotic regions, labelled viable cells became necrotic, and activity was slowly eliminated. With increasing proportions of necrosis during tumour growth, the rate of loss of activity of the whole tumour decreased. Loss of activity from viable tumour regions reflected cell death and exceeded the loss rates of the whole tumour by a factor of 2 to 3. The data show that loss of activity from the whole tumour results from a superposition of different elimination rates of viable and necrotic tumour regions and is not an immediate consequence of cell death in the course of undisturbed tumour development.