毕赤酵母表达重组人内皮抑素对小鼠肺腺癌LA795生长和转移的抑制作用

背景与目的：肿瘤的生长和转移有赖于新生血管的生成,内皮抑素能抑制肿瘤的血管生成。本研究旨在观察毕巴斯德酵母（pichia.pastoris.GS115)分泌表达的重组人内皮抑素（recombinant human endostatin,rhES)对小鼠肺腺癌LA795生长和转换的抑制作用。方法：挑取一株高效分泌表达rhES的毕赤巴斯德酵母菌株,利用甲醇进行大量诱导表达;用肝素亲和层析的方法纯化目的蛋白;将接种LA795肺腺癌细胞的T739小鼠随机分成两组,分别给予rhES和PBS皮下注射,每日1次,连续14天;观察两组小鼠肿瘤生长情况,测量肿瘤体积大小,并观察两组小鼠肿瘤肺部转移情况。结果：经甲醇诱导的毕赤巴斯德酵母菌株高效分泌表达了rhES;动物实验研究发现rhES能显著抑制小鼠肺腺癌LA795的生长,rhES治疗组肿瘤大小与对照组比较具有显著性差异（P＜0．001）,抑瘤率达到66．4％,并且有效抑制了肿瘤的肺部转移。结论：利用毕赤酵母作为宿主分泌表达的rhES具有良好的生物学活性,可显著抑制小鼠肺腺癌LA795的生长和转移。     

烟曲霉醇联合环磷酰胺对小鼠LA795肺腺癌转移的抑制作用

背景与目的:血管生成抑制剂联合化疗药物治疗肿瘤成为目前研究热点之一。本研究旨在观察烟曲霉醇(fumagillol,TNP-470)联合环磷酰胺(cyclophosphamide,CTX)对肺腺癌小鼠异体移植转移的协同抑制作用,并初步探讨TNP-470抑制肿瘤转移的相关机制。方法:将40只接种高转移性LA795肺腺癌细胞的T739裸小鼠随机分成5组:对照组、溶剂组、TNP-470组(30mg/kg)、CTX组(40mg/kg)、联合组(TNP-47030mg/kg CTX40mg/kg)。实验3周后,处死全部小鼠。剥离皮下肿瘤称瘤重并计算抑瘤率;取出双肺观察表面肿瘤转移情况,计算肿瘤肺转移发生率,计数各组小鼠肺表面转移结节数及计算出肺表面结节转移抑制率。免疫组化和图像分析系统检测皮下移植瘤中微血管密度(microvesseldensity,MVD)、P-选择素表达并定量分析。结果:联合组抑瘤率(81.5%)明显高于其他各组(P<0.01),Q值等于1.21,说明两药合用具有协同作用。与对照组(12.13±4.02)相比,联合组(1.75±1.71)、TNP-470组(4.75±3.34)、CTX组(8.50±2.67)肺表面转移结节数明显下降;同时TNP-470组和联合用药组皮下肿瘤内MVD、P-选择素表达与对照组相比均下降,差异有显著性(P<0.01),而CTX组对此则无明显影响。结论:TNP-470与CTX对LA795肺腺癌的肺结节转移具有协同抑制作用;TNP-470抑制LA795肺腺癌转移与其抑制肿瘤内P-选择素表达有关。     

小鼠肺腺癌（LA795）细胞球体移植于同基因鼠耳廓皮下后侵袭特性的研究

To evaluate invasion quantitatively and qualitatively in vivo, tumor cell aggregate invasion model was established. Mouse lung adenocarcinoma cell line (LA795) cells were allowed to form tumor cell aggregates after 3 days in a gyratory shaker culture system. LA795 cell aggregates, 0.3 mm in diameter, were transplanted subcutaneously to the auricles of syngenic T739 mice, which were then sacrificed at different intervals. Tumorigenicity was 100%. The process of invasion was divided into 5 stages: latent, proliferation, early invasion, mid and late invasion stages. In the mid and late invasion stages, cartilage and its capsule were invaded and progressively destroyed by tumor cells. Cartilage could be used as a criterion of judging invasion. Metastasis into the submaxillary lymph nodes (2/5) was found in the late invasion stage. The tumor cell aggregate invasion model is very useful in studying the invasion in vivo.     

MA737小鼠乳腺癌细胞的激光电泳行为

细胞表面负电荷增高是恶性细胞重要的膜特征之一。采用激光电泳散射技术测定了MA737小鼠乳腺癌及其正常乳腺细胞的电泳迁移率(EPM)结果示:MA737乳腺癌的多普勒频移高于正常乳腺细胞,二者频移的加权平均之比值为1.18,且前者较后者的谱峰展宽,同时发现,肿瘤细胞的C值大于正常细胞一个数量级,自相关函数的衰减速度也观察到明显不同。由此进一步表明,肿瘤与正常细胞膜表面特征的差异性,主要取决于细胞表面负电荷的影响。     

JCS对LA795肺腺癌小鼠移植瘤转移的抑制作用研究

目的对金刺参九正合剂（JCS）防治肺癌转移的能力及机制进行探讨。方法JCS/顺铂（DDP）作用于荷LA795高转移肺腺癌小鼠模型,观察药物对荷瘤小鼠抑瘤率、肺转移抑制率的影响,并应用免疫组化方法观察药物对实验小鼠皮下移植瘤体血管内皮生长因子（VEGF）、血管内皮细胞因子（CD34）、移植瘤黏附因子（CD44V6）、基质金属蛋白酶2（MMP2）、基质金属蛋白酶抑制酶（TIMP2）的表达及移植瘤微血管密度（MVD）变化的影响。结果JCS组/DDP＋JCS组瘤重抑制率、肺转移抑制率分别为18.2%、42.4%和60.6%、64.4%;MVD、VEGF、CD44V6、MMP2表达均明显低于其它组,TIMP2表达明显高于其它组（P〈0.01）。结论JCS对LA795高转移肺腺癌小鼠模型有较好抑制肿瘤生长、抑制转移的作用,其抑制基质降解及肿瘤血管生成,调节肿瘤转移相关黏附分子的表达可能是JCS抑制肿瘤转移的机制之一。     

β—胡萝卜素对TAⅡ小鼠移植性乳腺癌MA737影响的研究

观察β－胡萝卜素对移植性乳腺癌生长,荷瘤小鼠生存期及对ＣＭＦ化疗毒副作用的影响。方法采用ＴＡⅡ小鼠移植性乳腺癌模型,荷瘤小鼠给予ＢＣ和ＣＭＦ不同处理。结果（１）ＢＣ组、ＢＣ＋ＣＭＦ组平均每只小鼠生存时间较对照组显著延长,ＣＭＦ组小鼠因化疗毒性其生存时间较对照组明显缩短。（２）ＣＭＦ小鼠在实验７天内全部死亡外,ＢＣ组、ＢＣ＋ＣＭＦ组在实验不同时期肿瘤体积均明显小于对照组。（３）长期单独灌喂ＢＣ小鼠均     

内皮抑素对小鼠肺腺癌LA795生长和转移抑制作用的初步研究

目的：观察人内皮抑互对小鼠肺腺癌ＬＡ７９５生长和转移的抑制作用。方法：对重组人内皮抑素高效表达克隆ｐＣＸ的表达产物进行纯化,得到重组人内皮抑素（ｒｈＥＳ）。用亲和层析及胰弹性蛋白酶消化法从过期人血浆纯化得到人血管抑素（ｈＡＳ）。将接种ＬＡ７９５肺腺癌细胞的Ｔ７３９小鼠随机分成３组,分别给予ｒｈＥＳ,ｈＡＳ或等体积ＰＢＳ皮下注射,１次／日,共１４ｄ。观察３组肿瘤生长情况、肺湿重、肺表面转移结节数、动物生存期,分别进行ｑ检验。结果：ｒｈＥＳ组及ｈＡＳ组肿瘤生长缓慢,８ｄ后肿瘤逐渐回缩;肺湿重、肺表面转移结节数明显减少,动物生存期明显延长。结论：ｒｈＥＳ与ｈＡＳ均可明显抑制ＬＡ７９５所致的小鼠实验性肿瘤的生长与转移,延长动物的生存期。     

康莱特抗小鼠肺腺癌LA795肺转移相关基因表达谱的研究

目的 寻找康莱特抗肿瘤转移的表达差异基因。方法 以LA795小鼠肺腺癌肺转移组织，制备mRNA探针，应用含有4096条鼠基因的cDNA表达谱芯片，用ScanAr ray3000扫描仪扫描芯片荧光信号图像，分析肿瘤组织和康莱特治疗后肿瘤组织中差异表达基因。结果 发现在康莱特组和对照组存在差异表达的基因，其中重复3次均差异表达的基因有27条，其中表达增高的基因25条，表达降低的基因2条。其中12条是小鼠肌cDNA文库中的基因。经初步研究，发现6条可能与肿瘤发生、发展及转移相关的基因。结论 运用本基因表达谱芯片对基因表达谱进行分析，能够有效筛查出抗肺转移的相关差异基因。本组共发现6条与康莱特抗肿瘤转移相关的基因。     

IH小鼠全身热疗及热耐受实验

目的：研究全身热疗对机体的损伤、与热剂量的关系及全身热疗热耐受现象的量化。方法：分别以４０５℃、４１０℃、４１５℃、４２０℃、４２５℃、４３０℃,不同时间对ＮＩＨ小鼠进行加温治疗,观察其死亡情况。热耐受实验以４１５℃２５分钟作为引导加温,然后每隔２４小时按不同温度－时间进行治疗,记录其死亡率。结果：４１５℃、４２℃、４２５℃和４３℃不同加温时间的半数致死时间分别为４０８１ｍｉｎ、２２５２ｍｉｎ、１８２３ｍｉｎ及１３７８ｍｉｎ。热耐受研究证实热疗后２４小时最强,之后每２４小时消减近５０％,但４１５℃２４小时热耐受接近无穷大。４０５℃加热至３小时未见有死亡,４１℃产生最大死亡率为４０％。结论：小鼠全身热疗４１５℃以下约２小时后产生热耐受;２４小时产生最大热耐受,每２４小时约递减５０％。     

In vivo experiments, in vitro assays in experimental oncology: establishment of LA 795 Vv-Vt

In vivo experiment, in vitro assays (in vivo-in vitro system) is a kind of experimental technique which is different from both in vitro cell line and tumor line. The new model can be grown and studied either as an animal tumor or a cell culture. The specimen could be assayed for cell survival in vitro. This model can be used to study the response of malignant cells to the treatment in a precision and depth that is impossible in tumors grown in vivo. LA 795 Vv-Vt is the first in vivo-in vitro system developed in China. It was established with lung adenocarcinoma of T-739 mouse. The tumor cells could grow freely both in vivo and in vitro with a plating efficiency of 20%. Characteristics of LA 795 Vv-Vt tumor and cell in culture were described and compared. In order to estimate the radiation response of different doses, cell survival curves of tumors irradiated under oxic and hypoxic conditions were drawn and compared. The oxygen enhance ratio was 2.98. The experiments indicate that in vivo-in vitro system has a good dependent relation in dose and effect and is worth extensive application.     

TNP-470联合重组人内皮抑素抑制小鼠肺腺癌生长的研究

目的:观察TNP470和重组人内皮抑素（recombinant human endostatin,rhES）联合治疗对小鼠肺腺癌LA795肿瘤生长的抑制作用。方法: 用甲醇诱导能高效分泌表达重组人内皮抑素（rhES）的毕赤酵母菌株分泌表达rhES，将皮下接种LA795肺腺癌细胞的T739雄性近交系小鼠随机分成3组，每组各10只，分别给予PBS，rhES及TNP470+rhES皮下注射，每日1次，连续14 d；观察各组小鼠肿瘤生长情况，游标卡尺测量肿瘤体积大小。断颈处死小鼠，原位肿瘤免疫组化观察肿瘤内部微血管密度（microvessel density, MVD）。结果: 经甲醇诱导，毕赤酵母重组菌分泌表达rhES，并应用肝素亲和层析将其纯化；动物试验显示与PBS对照组比较，rhES治疗组及rhES＋TNP470治疗组均明显抑制小鼠肿瘤的生长（P<0.01），TNP470+rhES组与rhES组比较亦有显著性差异（P<0.01）。原位肿瘤免疫组化显示联合治疗组抑制血管生成更显著（P<0.01）。结论: TNP470联合rhES治疗对小鼠肺腺癌LA795生长的抑制效果比单独使用rhES的治疗效果更佳，具有良好的协同治疗作用     

LA795肺癌固化瘤苗抗肺癌LA795肺转移多种治癌疗法的实验比较

目的研究固化瘤苗免疫治疗癌症。方法用肺癌LA795制备成异构固化瘤苗（H一固化免疫因子）进行了横向比较的动物实验，观察其抗肺癌LA795肺克隆转移率。结果其抑癌率；对照组为O（平均肺转移156个克隆），化疗组（紫杉醇）96．09％，手术组76.9％，放疗组75．72％，固化瘤苗组76．15％，白介一2十手术65．32％。在这一系列数据中，固化瘤苗疗效可列入手术、化疗、放疗之中。固化瘤苗十手术与放疗十手术有效率达到96％土3。并从电镜中证实固化瘤苗免疫组均发现“凋亡小体”和一系列癌细胞自溶凋亡现象，呈现自主特异性细胞免疫效应。结论认为“固化瘤苗”的治疗效果已达到抗癌第四种方法“免疫治癌”的正式行列条件。如果实现“固化免疫外科”治癌．有可能使癌这一不治之症转为可治之症。     

INVASION OF SPHEROID OF MURINE LUNG ADENOCARCINOMA (LA_(795)) CELLS INTO EMBRYONIC CHICK HEART FRAGMENTS

An in vitro spheriod invasion model of tumor cell was established by using murine lung adenocarcinoma cell line (LA795) and precultured embryonic chick heart fragment (PHF). The spheroid of LA795 cells were prepared by incubating a suspension of trypsinized LA795 cells on a gyratory shaker. Spheroid aggregates of LA795 cells in diameter of 0. 2 mm were selected and confronted with PHF (diameter of 0. 4 mm) on semi- solid medium for 3 - 4 hours, then, individual confronting pain were transferred into fluid medium for further co-culture on gyratory shaker. After 1, 3, 5 and 7 days, multiple confronting pairs were processed for histological and ultrastructural study. The Invasive capacity and the invasion process of LA795 cells were examined and observed. The results demonstrated that LA795 cell line has a high capacity of invasion and high malignancy in vitro. This spheroid Invasion model is very useful for studying mechanism of Invasiveness of tumor cells in vitro.     

重组腺病毒rAdCDglyES对小鼠乳腺癌MA737放射效应的影响

目的:研究含CDgly ES双功能融合基因的重组腺病毒(rAdCDglyES)对肿瘤的直接、间接杀伤作用及其对放射的增敏作用。方法:建立MA737乳腺癌津白Ⅱ号小鼠荷瘤模型,按分组要求给予不同处理,设定多个观察指标对各组结果进行比较。结果:按分组要求处理后第25天,第1组瘤体达2.5cm×2.3cm;第6组肿瘤为1.4cm×1.2cm。第6组的中位生存期明显长于其余各组,P<0.01。第1组的凋亡指数较低,平均为2.61‰,而第6组的凋亡指数较高,平均为6.84‰,两组比较差异有统计学意义。第1组的微血管密度(MVD)值较高,为145.81,第6组的MVD值较低,为121.23,P<0.05。结论:含双功能融合基因的重组腺病毒在局部对肿瘤细胞具有直接杀伤作用,亦可抑制肿瘤新生血管的形成,对肿瘤细胞显示间接抑制、杀伤作用;显示该重组腺病毒能够和射线发生协同作用,增加局部放射对肿瘤的抑制作用。     

沉默TIPE2基因对T细胞共培养肺腺癌细胞LA795的免疫杀伤作用

目的 利用特异性小干扰RNA（small interfering RNA, siRNA）技术沉默T细胞TIPE2基因表达,体外观察转染T细胞对肺腺癌细胞LA795的免疫杀伤作用。方法 磁珠分选小鼠脾脏T细胞,筛选并验证能有效沉默T淋巴细胞TIPE2基因的siRNA序列,转染48 h后,利用酶联免疫吸附法（ELISA）比较各分组细胞上清液中IFN-γ分泌水平;转染72 h后,观察对比单纯T细胞（空白组）、阴性对照T细胞（阴性对照组）及转染T细胞（实验组）与小鼠肺腺癌细胞LA795共培养时肿瘤杀伤率。结果 我们筛选出一条能有效抑制TIPE2基因表达的siRNA序列,细胞转染率79.63%,蛋白表达抑制率达到79%,转染48 h后,检测实验组、阴性对照组、空白组细胞因子IFN-γ分泌水平分别为（678.96±26.91）pg/ml、（401.69±13.67）pg/ml、（387.03±15.14）pg/ml（P<0.001）;转染72 h后,与空白组及阴性对照组T细胞相比,在不同的效靶比水平（20:1、40:1、80:1）,转染T细胞杀瘤活性均更高。当效靶比为80:1时,实验组肿瘤细胞杀伤率达到（47.91±3.25）%。结论 利用特异性siRNA技术能有效沉默TIPE2基因,并促进抗肿瘤活性细胞因子IFN-γ分泌,增强T细胞对小鼠肺腺癌细胞LA795的体外免疫杀伤作用。     

Invasion of spheroid aggregate of mouse lung adenocarcinoma (LA 795) cells into embryonic chick heart fragments]

An in vitro invasion model of tumor cell spheroid aggregate was established by using mouse lung adenocarcinoma cell line (LA795) and precultured embryonic chick heart fragments (PHF). The spheroid aggregates of LA795 cells were prepared by incubating a suspension of trypsinized LA795 cells on a gyratory shaker. Spheroid aggregates of LA795 cells in diameter of 0.2 mm were selected and confronted with PHF (diameter of 0.4 mm) on semisolid medium for 3-4 hours, then, individual confronting pairs were transferred into fluid medium for further co-culture on gyratory shaker. After 1, 3, 5 and 7 days, multiplicated confronting pairs were processed for histological and ultrastructural study. The invasive capacity and the invasion process of LA795 cells were examined. The results demonstrated that LA795 cell line has a high capacity of invasion and malignancy in vitro. This spheroid invasion model is very useful for studying invasiveness of tumor cells in vitro.     

T739小鼠LA795肺腺癌皮下移植瘤氩氦刀冷冻消融前后基因表达谱芯片分析

[目的]用基因芯片技术分析、筛选氩氦冷冻消融前后肺腺癌相关基因谱的差异表达。[方法]建立T739小鼠LA795肺腺癌皮下移植瘤动物模型,行氩氦刀冷冻消融。分别抽提、纯化冷冻前后肿瘤组织mRNA,逆转录Cy3、Cy5标记cDNA探针,应用小鼠表达谱芯片CSC-ME-10,分析差异表达的基因。[结果]发现氩氦刀冷冻治疗前后T739小鼠LA795肺腺癌皮下移植瘤组织有显著性表达差异的基因共有26个,治疗后17条表达降低,9条表达增高。[结论]氩氦刀冷冻消融前后T739小鼠LA795肺腺癌皮下移植瘤组织部分功能基因存在着显著的表达差异,多数差异表达的基因与细胞生长因子调节、代谢酶类以及转录因子等相关。     

小剂量5-FU提高携带VEGF siRNA的腺病毒载体对肿瘤细胞的转导效率和基因治疗效果的研究

Background and Objective:Adenovirus vectors were widely used in gene therapy for tumors.We used adenovirus vector to transfer small interfering RNA(siRNA) against vascular epithelium growth factor A(VEGF-A) molecules to mouse lung adenoma LA795 cells and used low dose of chemotherapeutic drugs to further elevate the infection efficiency of adenovirus vector in and therapeutic effect of RNAi on tumor cells.Met hods:LA795 cells were infected by Ad/EGFP and treated with different dosages of gemcitabin,epirubic...     

Cardiac tumors of mice

Of the 7 primary tumors of the mouse heart, accessed at the Registry of Experimental Cancers, 3 were mesenchymal cell sarcomas, 3 were hemangioendotheliomas, and 1 was a mesenchymoma. The mesenchymal cell sarcomas metastasized via the bloodstream to lungs, kidneys, and spleen. Five of the tumors arose in mice treated with ionizing radiation. Previously, only 3 primary cardiac tumors of mice have been reported. Tumors of the heart as part of multifocal neoplasms of a single histologic type were observed in 3 mice; 2 of these tumors were type B reticulum cell sarcomas, and 1 was a reticulum cell sarcoma not otherwise specified. Of the metastatic tumors of the heart, 12 were from alveologenic carcinoma, squamous cell carcinoma, or carcinosarcoma of the lung, 4 were from sarcoma of the subcutaneous soft tissue, and 1 each was from a subcutaneous transplant of a malignant schwannoma and from an alveologenic tumor of the lung.     

Thermotolerance induced by heat and ethanol

The effect of ethanol and heat on the thermosensitivity of 3-day-old larvae of the fresh water snail Lymnaea stagnalis is investigated, especially with regard to the kinetics of thermotolerance, the effect on protein synthesis and the pattern of proteins synthesized. Both stress factors, in a mild dose, induce a state of thermotolerance with the following characteristics: (i) it is not accompanied by an enhanced synthesis of HSPs, (ii) it needs only 10-30 min to develop maximally, and (iii) it decays within 60-90 min after it has been triggered. At a higher dose both factors induce an enhanced synthesis of the HSP 65, HSP 70, and HSP 87, and also a more stable state of thermotolerance beside the thermotolerant state present shortly after the trigger. It appears that the synthesis of HSPs is enhanced only when the overall protein synthesis is depressed. The data are discussed in relation to the putative functions of heat shock proteins. It is suggested that the constitutive levels of the HSPs provide the rapid protection against heat. An induced level of HSPs is necessary for the extension of the thermotolerant state.