Dissecting the roles of E1A and E1B in adenoviral replication and RCAd-enhanced RDAd transduction efficacy on tumor cells

Oncolytic viruses have recently received widespread attention for their potential in innovative cancer therapy. Many telomerase promoter-regulated oncolytic adenoviral vectors retain E1A and E1B. However, the functions of E1A and E1B proteins in the oncolytic role of replication-competent adenovirus (RCAd) and RCAd enhanced transduction of replication defective adenoviruses (RDAd) have not been addressed well. In this study, we constructed viruses expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa. We then tested their roles in oncolysis and replication of RCAd as well as their roles in RCAd enhanced transfection rate and transgene expression of RDAd in various cancer cells in vitro and in xenografted human NCI-H460 tumors in nude mice. We demonstrated that RCAds expressing E1A alone and plus E1B-19 kDa exhibited an obvious ability in replication and oncolytic effects as well as enhanced RDAd replication and transgene expression, with the former showed more effective oncolysis, while the latter exhibited superior viral replication and transgene promotion activity. However, RCAd expressing both E1A and E1B-19 kDa/55 kDa was clearly worst in all these abilities. The effects of E1A and E1B observed through using RCAd were further validated by using plasmids expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa proteins. Our study provided evidence that E1A was essential for inducing replication and oncolytic effects of RCAd as well as RCAd enhanced RDAd transduction, and expression of E1B-19 kDa other than E1B-55 kDa could promote these effects. E1B-55 kDa is not necessary for the oncolytic effects of adenoviruses and somehow inhibits RCAd-mediated RDAd replication and transgene expression.     

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＳＯＦＨＵＭＡＮＩＬ－１５ＧＥＮＥＭＯＤＩＦＩＥＤＬＵＮＧＣＡＮＣＥＲＣＥＬＬＬＩＮＥＳｈｅｎＹｏｎｇｑｕａｎ１沈永泉ＣｕｉＬｉａｎｘｉａｎ２崔莲仙ＨｅＷｅｉ１何维ＸｕｅＬｉ１薛莉ＢａＤｅｎｉａｎ１巴德年１Ｉｎｓｔ...     

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.     

二氯化钴对腺病毒基因表达的影响

目的:研究二氯化钴(CoCl2)对缺氧反应元件调控E1AE1B表达的条件复制型腺病毒载体(Ad-5HRE-E1AE1B-RFP)和缺乏缺氧反应元件的复制缺陷型腺病毒载体(Ad-EGFP)在肿瘤细胞内表达和复制的影响. 方法:肿瘤细胞经不同浓度的CoCl2 处理后,Western 印迹法检测缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的表达;倒置荧光显微镜、FCM法及空斑形成实验等观察经CoCl2处理后,感染条件复制型腺病毒和(或)复制缺陷型腺病毒的肿瘤细胞中外源基因的表达水平和病毒复制情况;小动物成像仪观察缺氧调控的条件复制型腺病毒在肿瘤内提高复制缺陷型腺病毒表达的效率. 结果:适当浓度的CoCl2 (0.4和0.08 μg/mL) 能使胃癌细胞株(SGC7901)中HIF-1α蛋白稳定表达并积聚,可较好地模拟缺氧状态;在0.4 μg/mL CoCl2作用下,Ad-5HRE-E1AE1B-RFP对肿瘤细胞的感染效率明显提高,表现为外源基因RFP阳性表达的百分率和荧光强度均明显提高,但空斑形成实验显示Ad-5HRE-E1AE1B-RFP并没有复制.0.4 μg/mL CoCl2也能提高其他非缺氧调控的复制缺陷型腺病毒如Ad-EGFP、Ad-Luc的感染效率和表达水平; Ad-5HRE-E1AE1B-RFP与复制缺陷型腺病毒Ad-Luc联合注射裸鼠肿瘤能显著提高Ad-Luc的表达.结论:CoCl2 能明显提高腺病毒的基因表达水平,其作用机制不仅与CoCl2诱导缺氧有关,也可能与影响基因转录有关.     

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...     

小鼠肺癌B7疫苗细胞FLB2C诱导的抗肿瘤免疫应答

目的：研究小鼠肺癌细胞与活化B淋巴细胞融合的疫苗细胞FLB2c诱导细胞免疫反应情况。方法：用母本细胞LA795作对照，采用体外混合淋巴细胞培养，观察FLB2c细胞刺激T细胞增殖情况，通过CTLL细胞MTT法测定FLB2c刺激T细胞产生分泌IL－2的作用，用FLB2c免疫小鼠，观察疫苗体内诱导小鼠CTL活性的能力。结果：FLB2c细胞在体外刺激T细胞增殖的作用明显比LA795强；FLB2c能刺激T细胞分泌IL－2，而LA795则不能；FLB2c细胞在体内能诱导CTL活性，其诱导CTL在体外对FLB2c细胞和LA795的杀伤率分别为34％－25．3％，而LA795诱导的CTL对FLB2c和LA795杀伤率分别为10．5％和12．25％，两者的杀伤率有显著性差异。结论：FLB2c细胞在体内外均能刺激T细胞免疫应答，其以能力明显强于未融合的LA795小鼠肺癌细胞，将其作为疫苗细胞将有可能通过提高机体免疫应答而达到治疗肺癌的效果。本研究为进一步探讨该疫苗的应用价值奠定了免疫学基础。     

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.     

Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant

Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.     

依维莫司负调控mTOR信号通路对膀胱癌BTT739细胞增殖和迁移的抑制作用及机制研究

目的:探索mTOR抑制剂依维莫司(RAD001)对膀胱癌BTT739细胞在体外和小鼠体内增殖和迁移的抑制作用及其作用机制,为依维莫司在临床上治疗膀胱癌提供理论依据。方法:体外培养小鼠膀胱癌BTT739细胞,待其生长至对数期后用梯度浓度的RAD001（0 nmol/L、5 nmol/L、10 nmol/L和20 nmol/L）干预24 h,用MTT法检测细胞增殖的变化;Tranwell实验检测RAD001对BTT739细胞迁移的影响;ELISA试剂盒检测细胞分泌蛋白E-钙黏蛋白表达的变化。Western blot检测Akt、mTOR、4EBP和PTEN蛋白的表达,RT-PCR检测mTOR mRNA的表达。将BTT739细胞接种于昆明鼠皮下,待成瘤后,用依维莫司干预14天,取小鼠肿瘤组织检测E-钙黏蛋白及AKT、mTOR、4EBP和PTEN蛋白表达的情况。结果:体外实验显示RAD001可以抑制BTT739细胞的增殖和迁移并与剂量有关（P＜0.05）。还可以增加E-钙黏蛋白的含量,抑制癌细胞的转移。实验证明RAD001在体外抑制Akt、mTOR、4EBP的表达,促进肿瘤抑制因子PTEN的表达（P＜0.05）。将BTT739细胞接种于小鼠皮下后发现,RAD001在小鼠体内亦可以抑制病变情况,促进PTEN和E-钙黏蛋白的表达,抑制Akt、mTOR和4EBP的表达。结论:mTOR抑制剂依维莫司通过抑制mTOR信号通路抑制膀胱癌细胞BTT739的增殖、迁移和侵袭。     

Suppression of metastatic hemangiosarcoma by a parvovirus MVMp vector transducing the IP-10 chemokine into immunocompetent mice

We have previously shown that the growth of human tumor xenografts in immunodeficient mice can be efficiently suppressed upon infection with the autonomous parvovirus H-1 or with cytokine-transducing derivatives thereof. To further evaluate the benefits of implementing parvoviruses in cancer gene therapy, we have created a new recombinant vector, MVMp/IP-10, transducing the immunoactive, antiangiogenic chemokine IP-10, and used this virus to treat syngeneic tumors grown in immunocompetent mice. Intratumoral/intraperitoneal administration of only 3 x 10(7) replication units of MVMp/IP-10 per animal strongly inhibited the progression of established H5V cell-induced vascular tumors, a highly malignant mouse model for human cavernous hemangioma and Kaposi's sarcoma. Retardation of recurrent tumor growth and suppression of life-threatening metastatic dissemination to internal organs were accompanied by a striking delay in hemangioma-associated mortality. Parental MVMp did not have a significant effect under these conditions up to the dose of 10(10) infectious units/animal, but had strong antihemangiosarcoma activity when used to infect H5V cells ex vivo prior to implantation. In all cases, virus therapy was very well tolerated. Virus-induced suppression of hemangiosarcoma was dependent on host T cells and associated with intratumoral persistence of IFN gamma-expressing cytotoxic lymphocytes, and led to the reduced expression of hepatic plasminogen activator inhibitor-1 (PAI-1), a metastasis-linked marker. This proof of principle study demonstrates that MVMp/IP-10 can aid the treatment of vascular tumors and that autonomous parvovirus-based vectors can be considered potent tools for cancer gene therapy purposes.     

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.     

Formosanin C-inhibited pulmonary metastasis through repression of matrix metalloproteinases on mouse lung adenocarcinoma

Formosanin C (FC) isolated from Rhizoma Paridis, showed pro-apoptosis and immunoregulation with antitumor activity in cultured cells and animal systems. However, there is no report about its anti-metastatic effect on cancer cells. This research used the wound healing and the migration assay to detect the anti-invasive effect of FC on LA795 cells. Through the gelatin zymography assay and immunofluorescence analysis, FC showed suppression of enzyme activity of MMP-2 and MMP-9, and protein expression of MMP-1, -2, -3, -9 and -14 excreted from LA795 cells. Finally, FC exhibited much more effective inhibition of tumor growth and pulmonary metastasis in T739 mice than cisplatin did. Overall, the strong inhibition of MMP expression by FC might provide a potential therapeutic modality for lung tumors.     

Establishment, characteristics, and utilization of a new in vivo-in vitro system

Radiotherapy and chemotherapy are two important methods for malignant tumor treatment. To research radiobiological response in therapy, we have established a better experimental method in contrast to the traditional ones such as TCD50, regrowth delay, cell survival curve, etc, all with their limitations. A new mouse tumor in vivo-in vitro system LA795 Vv-Vt has been developed for studies on radiobiology. Such a system could be used to study the in vivo response of a solid tumor by the in vitro cloning assay. For the purpose of increasing the PE in vitro, LA795 Vv-Vt tumor line was purified through culturing the cells as a clonogenic spheroid. The spheroids were then injected into the flank of mouse subcutaneously for tumor growth. The in vivo-in vitro system LA795 Vv-Vt is an excellent model dissecting and analyzing the various factors which affect tumor development and determine the response of tumor to specific agent and regimens.     

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。     

Host cell range of adult T-cell leukemia virus. I. Viral infectivity and binding to various cells as detected by flow cytometry

Adult T-cell leukemia virus is the member of a human type-C retrovirus family (HTLV) found to be associated with adult T-cell leukemia (ATL) in Japan. In our study, HTLV was isolated from the MT-2 cell line, purified on sucrose gradient and labelled with fluorescein-isothiocyanate (FITC-HTLV). The protein pattern of the virus was determined by SDS-gel electrophoresis and assured by Western blotting using ATL patient serum. Fresh human lymphocytes, separated B and T cells, mouse and rabbit lymphocytes, mouse fibroblasts, and 13 different tumor cell lines were tested in parallel for binding of FITC-HTLV and infectability by the virus. Virus binding to cell receptors was assayed by flow cytometry. Successful infection was monitored by following the expression of HTLV-determined antigen (HTLA). Most of the cells bound FITC-HTLV at levels ranging from 5% to 130% of the MT-2 cell binding. Only fresh human T, mouse and rabbit lymphocytes were infectable by cell-free virus preparations. The results demonstrate that HTLV receptors are present on different types of cells of both human and animal origin, and that infection by the virus is restricted to fewer host cells but not limited to a specific class of human lymphocytes.     

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.     

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。     

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。     

An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression

Over the past decades, much has been learnt about the genes that contribute to oncogenic transformation of primary cells in vitro. However, much less is known about the genes that contribute to the later stages of tumor progression, in which cells of ever increasing malignancy arise through clonal selection in vivo. To search for genes that confer a tumor progression phenotype in vivo, we have used a functional genetic approach. We used adenovirus-transformed mouse embryo fibroblasts, which are tumorigenic in immunodeficient nude mice, but not in immunocompetent mice, due to strong cytotoxic T-cell-mediated immune rejection. We infected these cells in vitro with several high-complexity retroviral cDNA expression libraries and selected rare variants that formed tumors in immunocompetent mice. Using this approach, we identify here the TRK-T3 oncogene as a tumor progression gene. TRK-T3 does not inhibit T-cell reactivity towards the tumor cells. Instead, we find that cells expressing TRK-T3 enhances in vivo growth rate, most likely by stimulating anchorage-independent proliferation in growth factor-limiting conditions. Our data indicate that cDNA expression libraries can be used to identify tumor progression genes in vivo that cannot be readily identified using in vitro cell culture systems.     

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。