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.     

抗腺病毒载体单克隆抗体的制备及其初步鉴定

Objective:To prepare and identify monoclonal antibodies(McAbs)against the capsid proteins of adenovirus vector.Methods:BALB/c mice were immunized with a mixture of the purified adenovirus vector(Adv)and Al(OH)3.McAbs were produced using cell fusion technique in a conventional way.The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay(ELISA),immunocytochemical staining and Western blotting. Results:Six strains of hybridoma cells(A4H11,A8C7,F1H5,G1D2,G4E3 and H2G8)that can stably secrete the IgG1 McAb against Adv were obtained.After 3 months subculture and low concentration of serum adapting culture,six strains retained their stability to secrete McAb.The ascites titers were between 1:106 and 1:108.Western blot analysis demonstrated that all the McAbs reacted with one protein(about 114 kDa)which is present in wild type 3 adenovirus(wtAd3),wild type 5 adenovirus (wtAd5),wild type 7 adenovirus(wtAd7)and adenovirus vector.Conclusion:Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector,and provided the substantial foundation of preclinical research of adenovirus vectors.     

Effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes via recombinant adenovirus

　Objective To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs. In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance (MDR).     

ISOLATION OF HUMAN TRANSCRIPTS EXPRESSED IN 16HBE CELLS RELATED TO CHLOROPHYLLIN ANTITRANSFORMING ACTIVITY AGAINST ANTI-BPDE BY cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

Objective: To analyze the differentially expressed eDNA sequences related to ehlorophyllin (CHL) mediated inhibition of malignant transformation of human bronchial epithelial cell line (16HBE). Methods: 16HBE cells treated with chlorophyllin and anti-BPDE were conducted as tester, 16HBE cells treated only with anti-BPDE were conducted as driver,and eDNA representational difference analysis (eDNA RDA) was used to compare the differential gene expression between the two kinds of cells. The eDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA was sequenced and compared with database in GenBank by BLASTN. Results: Among the 5 cloned eDNA sequences, three were novel and were registered in dbEST database, two showed sequence homology to alpha-enolase and a newly found gene ribosomal protein S18/S6-1ike. Conclusion: These 5 eDNA sequences might play important roles in antitransforming effect of chlorophyllin.     

CEA siRNA载体的构建和转染人食管癌EC9706细胞的沉默作用

Objective: To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods: Two pairs of oligonucleotide sequences were designed and synthesized according to the encoding sequence of mRNA of CEA. The annealed oligonucleotide frag-ments were cloned into pRNAT-U6.2 expression vector and identified by sequencing. The recombinant plasmid pRNAT-U6.2-CEA was transfected into EC9706 cells. The expression of CEA in the stable transfected cells was assayed by real time PCR and Western blot. Results: DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.2 vector, and CEA expression in the transfected cells was down-regulated significantly by pRNAT-U6.2-CEA at both the mRNA and protein levels. Conclusion: The siRNA expression vector of CEA is successfully constructed and inhibits CEA expression in EC9706 cells. This facilitates further studies of the function of CEA at the molecular level.     

Anti-tumor Activities of Novel Estrogen Compound 17aα-D-Homo-Ethynylestradiol-3-Acetate

Objective:To study the anti-tumor activities of novel estrogen compound 17a α-D-homo-ethvnvlestradiol-3-acetate in vitro and in vivo. Methods:In vitro anti-tumor activity was assayed in adenoma cells A549 and human liver cancer cells Bel-7402 using MTT method,and half-inhibitory concentration (IC50)were observed. In vivo the pulmonary adenoma LA795 cells was selected and the conventional assay method of anti-tumor activity was employed.5,7.5,10 mg/kg of 17a α-D-homo-ethynylestradiol-3-acetate was administered by i.P., and tumor-inhibitory rate, thymus and spleen indexes,bone marrow cells(BMC)were observed. Results:IC50 of 17a α-D-homo-ethynylestradiol-3-acetate in vitro for A549 and Bel-7402 cells were 12.28 μg/ml and 17.79 μg/ml, respectively.In vivo the highest tumor-inhibitory rates for LA795 was 60.0%(P＜0.01).The drug had hardly any side-effect in spleen indexes,thymus indexes,and BMC compared with control mice. Nevertheless,compared with the positive control drug cyclophosphamide(CY),thymus and spleen indexes,BMC showed obvious diffefences(P＜0.01). Conclusion:17a α-D-homo-ethynylestradiol-3-acetate has obvious anti-tumor activities in vitro and in vivo with low side-effect, thus worth further investigation.     

Preparation and identification of monoclona antibodies against the adenovirus vector

Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.     

沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

TβR-Ⅱ-RANTES融合基因重组腺病毒的抗肿瘤作用

目的构建表达融合基因TGF-βⅡ型受体(TβR-Ⅱ)胞外区及活化T细胞表达和分泌的调节因子(RANTES)重组腺病毒载体,并观察其抗肿瘤作用。方法RT-PCR扩增小鼠TβR-Ⅱ胞外区和RANTES基因,重叠PCR扩增融合基因TβR-Ⅱ胞外区-RANTES。采用adMax adenovjrus vector creation试剂盒构建表达融合基因重组腺病毒。体外感染小鼠肺腺痛LA795细胞系,绘制细胞生长曲线。采用Western blot法检测感染后细胞融合基因的表达;酶联免疫吸附试验(FLISA)法检测其培养上清的蛋白含量;Annexin V-FITC法检测感染后细胞的凋亡;并观察感染后细胞培养上清对小鼠脾细胞的趋化作用。将感染后的细胞(1×105个)接种于T739小鼠,观察成瘤时间和生存时间;1×1010pfu的重组腺病毒局部注射荷瘤小鼠,观察肿瘤的大小变化,并统计肿瘤的重量和计算抑瘤率。结果经测序证实,RT-PCR正确扩增小鼠TβR-Ⅱ胞外区和RANTFS基因,重叠PCR正确扩增融合基因TβR-Ⅱ胞外区-RANTES。重组质粒pDC316-融合基因经酶切鉴定正确,与pJM17双质粒共转染获得表达融合基因重组腺病毒,病毒滴度为8×1010pfu/ml。Western blot结果表明,感染后的LA795细胞有融合蛋白的特异性条带出现;培养上清中,游离TGF-β1的水平显菩减低,而RANTES的水平显著升高。感染融合基因重组腺病毒的细胞生长速度明显减低,凋亡率为16.9%;培养上清可趋化小鼠脾细胞,与对照组之间差异均有统计学意义。感染融合基因重组腺病毒组与对照组相比,体内成瘤时间和生存时间明显延长;局部注射融合基因重组腺病毒可显著抑制肿瘤的生长,抑瘤率为37.6%。结论成功构建表达融合基因TβR-Ⅱ胞外区-RANTES重组腺病毒可有效结合TGF-β1,显著逆转TGF-β介导的免疫抑制状态,高水平表达RANTES强化肿瘤局部的免疫功能,具有显著的抗肿瘤作用。     

A novel immunocompetent murine tumor model for the evaluation of RCAd-enhanced RDAd transduction efficacy

Low gene transfer rate in tumors, high dose-induced acute inflammatory response, and lack of an immunocompetent preclinical animal model to accurately reflect the therapeutic efficacy are prominent reasons for the lack of clinical success of adenoviral (Ad) vectors. In this study, we tested whether human replication-competent adenovirus (RCAd) can replicate in T739 mouse bladder transitional tumor cells (BTT) and lung adenocarcinoma cells (LA795), and whether RCAd can enhance the transduction rate and transgene expression of human replication defective adenoviruses (RDAd) in these tumor cells in vitro and in vivo. We demonstrated that human RCAd exhibited good infectability and cytopathologic effects in mouse BTT and LA795 cells, which was comparable to that in A549 and NCIH460 human tumor cells. In contrast, no infectability and cytopathologic effects were observed in other three mouse tumor cells such as 4T1, B16, and Lewis cells. The combined use of RCAd with RDAd significantly enhanced RDAd transduction efficiency in BTT and LA795 tumor cells in vitro and in vivo. When BTT and LA795 cells were co-infected with RDAd Ad-EGFP and RCAd, a large amount of E1a expression and 2-3 orders of increases in Ad-EGFP genomic DNA were observed. In contrast, the expression of the late gene Hexon is very low, which may explain ineffective packaging of viral particles. In conclusion, our study provided a novel immunocompetent animal model which is useful for evaluating RCAd infectability, cytopathy, and replication. The combined use of RCAd and RDAd provided a new solution for cancer gene therapy.     

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.     

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

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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。     

含HSV1-TK基因重组腺病毒的构建及对小鼠肺癌的抑制作用

目的　观察含单纯疱疹病毒 1型胸苷激酶 (HSV1- TK)基因和 CMV启动子的重组腺病毒Ad E1 CMVHSV1- TK(Ad TK)对 L795小鼠肺癌细胞和 T739近交纯系小鼠肺癌的抑制作用。方法　采用同源重组的方法在人胚肾 2 93细胞中构建重组腺病毒 Ad TK ,用 PCR方法鉴定 ;TCID50 (5 0 %组织细胞感染量 )方法滴定病毒 ;将荷瘤小鼠分为 DMEM对照组 ,Ad L ac Z对照组 ,Ad TK实验一组 ,Ad TK实验二组 ;连续观察各组小鼠肿瘤的生长速度以及存活期。结果　 PCR证实了 TK基因的导入 ;动物实验结果显示实验组小鼠瘤体的生长速度低于对照组 ,且生存期长于对照组 ,均有统计学差异。肿瘤组织病理切片显示对照组肿瘤组织比实验组生长旺盛。结论　可在人胚肾 2 93细胞中同源重组构建重组腺病毒 Ad TK,且能达到治疗有效滴度 ;Ad TK/ GCV系统对小鼠肺癌具有明显的治疗效果。     

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.     

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。     

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。