抗人乳头瘤病毒16型E6基因核酶对宫颈癌CaSKi细胞生长和端粒酶活性的影响

目的研究抗HPV16E6核酶(Ribozyme)对宫颈癌CaSKi细胞生长和端粒酶活性的影响。方法以脂质体法将抗HPV16E6-Ribozyme、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R和CaSKi-P细胞。点杂交检测核酶在细胞中的表达,northern杂交检测三种细胞中E6基因的表达;细胞生长曲线检测细胞生长的改变;TRAP-Elisa法检测端粒酶活性。结果点杂交证实核酶能在CaSKi-R细胞中稳定表达,northern杂交证实CaSKi-R中表达E6较CaSKi-P和CaSKi明显降低。CaSKi-R细胞生长速度较CaSKi-P和CaSKi明显减慢(P<0.01);CaSKi,CaSKi-P和CaSKi-R三种细胞的端粒酶活性分别为0.89±0.14,0.90±0.11,0.36±0.06,转染了抗HPV16E6核酶的CaSKi-R的端粒酶活性的抑制率为59.55%。经统计学处理,CaSKi-R的端粒酶活性较CaSKi和CaSKi-P细胞明显下降(P<0.01)。结论转染抗HPV16E6-Ribozyme的CaSKi-R细胞出现一定程度的生长抑制,端粒酶活性较前明显下降。

脑胶质瘤辐射抗性与逆转问题研究进展

胶质瘤是最常见的脑肿瘤,也是辐射抗性较强的肿瘤组织。近年来,对电离辐射诱导细胞凋亡分子机制的重视,探讨胶质瘤细胞的辐射抗性机制,寻找可逆转抗性基因,提高对肿瘤细胞的辐射敏感性,从而提高治疗的效果。本工作就胶质瘤细胞辐射抗性相关的基因、相关的酶、细胞外环境的作用及辐射抗性逆转的几个问题展开了讨论。

基于PI3K/AKT信号通路探讨补肾壮骨汤对骨关节炎防治作用的实验研究

目的:基于PI3K/AKT信号通路探讨补肾壮骨汤对骨关节炎防治作用的机制。方法:取雄性SD大鼠30只,随机分为对照组、模型组和补肾壮骨组。其中对照组和模型组均给予等体积0.9%氯化钠溶液灌胃;而补肾壮骨组给予补肾壮骨汤灌胃,连续给药4周。干预完成后处死大鼠,采集血液并取关节软骨组织。采用酶联免疫吸附测定(ELISA)检测血清中白细胞介素(IL)-1β、IL-6和IL-10的含量。采用实时定量聚合酶链反应(RT-qPCR)检测磷脂酰肌醇3激酶(PI3K)、丝氨酸-苏氨酸激酶(AKT)和哺乳动物雷帕霉素靶蛋白(mTOR)基因表达;采用Western blot检测抗凋亡基因B淋巴细胞瘤-2(Bcl-2)和Survivin蛋白、促凋亡基因Bcl-2相关X蛋白(Bax)表达。结果:与对照组比较,模型组的软骨细胞活性明显降低;IL-1β和IL-6的含量均明显升高,而IL-10的含量显著下降;PI3K、AKT和mTOR基因和蛋白表达水平均显著降低;Bcl-2和Survivin蛋白表达增加,Bax蛋白表达降低(均P<0.05);而与模型组比较,补肾壮骨组的软骨细胞活性明显增加;IL-1β和IL-6的含量明显下降,而IL-10的含量显著上升;PI3K、AKT和mTOR基因和蛋白表达水平均显著升高;Bcl-2和Survivin蛋白表达下降,Bax蛋白表达上升(均P<0.05)。结论:补肾壮骨汤有助于促进OA软骨细胞增殖、抑制软骨细胞凋亡,发挥抗炎作用,其机制可能与调控PI3K/AKT通路,进而介导软骨细胞增殖凋亡和抗炎作用有关。

乳腺癌BAG-1基因表达与表皮生长因子受体表达的相关性

目的探讨乳腺癌BAG-1基因表达与表皮生长因子受体(EGFR)表达的相关性。方法培养乳腺癌细胞株,采用实时聚合酶链反应技术检测EGFR(+)和EGFR(-)细胞株中BAG-1 m RNA的表达情况,West blot法检测其蛋白水平。分析BAG-1基因表达与EGFR表达的相关性。结果 MDA-MB-231、T47D细胞株EGFR m RNA和EGFR蛋白阳性表达,MCF-7、SLBP3细胞株EGFR m RNA和EGFR蛋白阴性表达。T47D细胞株BAG-1 m RNA的表达水平最高[(29.6±0.4)×10-4],其次为MDA-MB-231细胞株[(6.9±0.3)×10-4],两者的表达水平均显著高于MCF-7细胞株[(4.3±0.5)×10-4]、SKBR3细胞株[(0.3±0.1)×10-4],差异的有高度统计学意义(P<0.01)。结论BAG-1表达与EGFR存在相关性,可作为乳腺癌靶向治疗的潜在靶点。

The in vitro Anti Hptocarcinoma Effects of Human Interleukin-12 from Transgenic Potato

[Objective]The aim was to examine the anti heptocarcinoma effects of human interleukin-12（hIL-12） from transgenic potatoes.[Method]Human heptocarcinoma cell line HepG22.2.15 was cocultured with human peripherial blood monocyte（PBMC）.Human IL-12 extracted from its transgenic patatoes was introduced to this coculture system.MTT method and laser confocal microscope were then employed to evaluate its survival rates and morphological changes of HepG22.2.15 cell line.[Result]The HepG22.2.15 survival rate of the plant produced hIL-12 treated group was significantly lower than that of the wild type control,while similar to that of commercial purified recombinant hIL-12 group.As indicated by AnnexinV/PI double staining laser confocal microscope,cocultured heptocarcinoma cells showed the typical early and final phase apoptotic morphological characteristics 48 hours after 2 × 10-4 mg/L potato secreted hIL-12 treatment.[Conclusion] These results demonstrated that Heptocarcinoma HepG22.2.15 cell growth could be actively inhibited by the transgenic potato expressed hIL-12 in vitro.

Expression and Mutation of MAGE-A3 mRNA in Lung Cancer Tissues

Objective： To inwvetigate the expression of MAGE-A3 mRNA in tissue samples derived from lung cancers and to discuss the possibility of using MAGE-A3 antigens as a new peptide vaccine for inunotherapy for lung cancers. Methods： Tumor tissue samples of lung cancers and paired non-tumor tissues of the lung were obtaimed from 31 lung cancer patients. Total RNA was extracted and cDNA was synthesized. Nested polymernse chain reaction amplification using MAGE-A3 specific primer was performed to detect the expression of MAGE-A3. The 10 clones of 5 samples of MAGE-A3 mRNA positive PCR products were DNA sequenced by using DNAs sequencer （PE-377）. Results： Of 31 lung cancers, 26 （83.9%） expressed MACE-A3 mRNA. The expression of MAGE-A3 gene was not detectable in the adjacent lung tissues. The DNA sequencing confirmed that the target gene fragment in all 5 samples of PCR products was MACE-A3 cDNA. Point nmtations occurred in 4 samples （8 clones） detected （C^2773→T^2773; G^2807→A^2807） resulting in alternation of amino acid residue in one position （E^143→K）. Conclusion： （1） The MAGE-A3 gene was expressed exclusively in tumor tissues of the patients with lung cancer in China. This tumor rejection antigen may have potential to be used as a new peptide vaccine for immunotherapy for lung eancers. （2） There are two point mutations of MAGE-A3 gene sequence in some Chinese lung cancer patients.

Obtaining High Pest_resistant Transgenic Upland Cotton Cultivars Carrying cry1Ac3 Gene Driven by Chimeric OM Promoter

Hypocotyl segments from aseptic seedlings of two important cultivars of upland cotton ( Gossypium hirsutum L.) in Northwest China, 'Xinluzao_1', 'Jinmian_7', 'Jinmian_12' and 'Jihe_321' were transformed respectively by two efficient plant expression plasmids pBinMoBc and pBinoBc via Agrobacterium tumefaciens . In pBinMoBc, cry 1Ac3 gene, which encodes the Bt toxin, is under the control of chimeric OM promoter. In pBinoBc, it is under control of CaMV 35S promoter. After co_cultivation with Agrobacterium tumefimpfaciens LBA4404 (containing pBinMoBc or pBinoBc), kanamycin_resistant selection, somatic embryos were induced and regenerated plants were obtained. Then the regenerated plantlets were grafted to untransformed stocks in greenhouse to produce descendants. The integration of cry 1Ac3 gene and its expression in T 2 generation of transgenic cotton plants were confirmed by Southern hybridization and Western blotting. The analyses of insect bioassay indicated that the transgenic plants of both constructions have significant resistance to the larvae of cotton bollworm ( Heliothis armigera ) and that cry 1Ac3 gene driven by chimeric OM promoter could endue T 2 generation cotton with high pest_resistant ability, implicating that it has a profound application in genetic engineering to breed new pest_resistant cotton varieties.

Differential Expression of PKC Isoforms and Their Tumoricidal Activity in Two Macrophage Cell Lines: Involvement of Nitric Oxide-dependent Mechanisms

Objective: To investigate the role of PKC isoforms in the regulation of LPS-triggered tumoricidal activity in macrophages and further elucidate its signal mechanisms. Methods: Two macrophage cell lines (P388D1 and RAW264.7) were stimulated by LPS alone, or with long-term of PMA pretreatment. Then cytotoxicities to P815 cells (by MTT assay) and IL-1, TNF- (by ELISA) and nitric oxide (NO) production (by Griess reagent) in supernatants were measured. Western blot for PKC isoforms after long-term PMA pretreatment was analyzed. Results: RAW264.7 cells were stimulated with LPS to kill target tumor cells P815, whereas P388D1 cells failed to develop such an ability. Down-regulation of PKC isoforms by chronic treatment with PMA significantly inhibited the LPS-induced cytotoxicity in RAW264.7 cells. In unstimulated state, Western blotting with rabbit antiserum specific for the PKC, 1, 2, or showed all 5 isoforms were detected in P388D1 cells, while only PKC, PKC1 and PKC were detected in RAW264.7 cells. Exposure of the cells to long-term of PMA treatment significantly down-regulated the expression of PKC, PKC1 and PKC in RAW264.7 cells. But in P388D1 cells, although PKC, PKC and PKC were down-regulated, the expression of PKC1 and PKC2 could not be regulated. Comparing with LPS-induced IL-1, TNF- and NO production by the two macrophage cell lines, P388D1 failed to produce NO. In RAW264.7 cells, LPS-induced NO production and antitumor activity was attenuated by the addition of L-NAME, an iNOS inhibitor. Conclusion: The results indicated a critical role of PKC in LPS-induced antitumor activity and this cytotoxicity is mainly due to PKC- mediated NO production by RAW264.7 cells, but not a direct cytotoxic activity.

Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

Monoclonal antibodies as therapeutic agents in oncology and antibody gene therapy

Antibodies as therapeutic agents are mostly used in oncology, as illustrated by their applications in lymphoma, breast cancer or colorectal cancer. This review provides a brief historical sketch of the development of monoclonal antibodies for cancer treatment and summarizes the most significant clinical data for the best-established reagents to date. It also discusses strategies to improve the anti-tumor efficacy of antibody therapy, including antibody gene therapy and exploitation of bone marrow derived primary mesenchymal stem cells as the antibody gene transporter.

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185^(c-erbB-2)

The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p!85. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.

Activation of STAT3 signaling in human stomach adenocarcinoma drug-resistant cell line and its relationship with expression of vascular endothelial growth factor

AIM: To investigate the difference in activation of STAT3 signaling between two human stomach adenocarcinoma cell lines: 5-fluorouracil resistant cell line and its parental cell line, and to evaluate its relationship with the expression of vascular endothelial growth factor (VEGF). METHODS: Western blot and electrophoretic mobility shift assay (EMSA) were used to detect the expression of phospho-STAT3 protein and constitutive activation of STAT3 in two human stomach adenocarcinoma cell lines, 5-fluorouracil resistant cell line SGC7901/R and its parental cell line SGC7901, respectively. The mRNA expression of VEGF was analysed by semi-quantitative RT-PCR. The expressive intensity of VEGF protein was measured by immunocytochemistry. RESULTS: The expressions of phospho-STATS protein and constitutive activation of STAT3 between two human stomach adenocarcinoma cell lines were different. Compared with the parental cell line SGC7901, the STAT3DNA binding activity and the expressive intensity of phospho-STAT3 protein were lower in the drug-resistant cell line SGC7901/R. The expression levels of VEGF mRNA and its encoded protein were also decreased in drugresistant cell line. CONCLUSION: Over-expression of VEGF may be correlated with elevated STAT3 activation in parental cell line. Lower VEGF expression may be correlated with decreased STAT3 activation in resistant cell line, which may have resulted from negative feedback regulation of STAT signaling.

Anti-tumor effect of CTLs activated by dendritic cells pulsed with K-ras mutant peptide and whole tumor antigen on pancreatic cancer

Objective： We studied the role of specific cytotoxic T lymphocytes （CTLs） activated by dendritic cells （DCs） presenting cationic nanoparticles with the K-ras （12-Val） mutant peptide and whole tumor antigen in the killing of different pancreatic cancer cell lines in vitro and in vitro. Methods： Peripheral blood DCs were induced by rhGM-CSF and IL-4 and cultured. DCs were sensitized by whole antigen of a pancreatic cancer cell line （PANC-1） with expression of K-ras mutant, K-ras mutant peptide （K-ras＋peptide） and cationic nanoparticles with K-ras mutant peptide （K-ras＋peptide-CNP）, respectively. Cell surface markers were measured by flow cytometry. Lymphocyte proliferation was detected by the 3H-TdR test, and ELISAwas performed to detect IFN-y secretion. 125I-UdR was used to measure the killing effect of CTLs. We also evaluated the antitumor activity of CTLs in vivo in a tumor-bearing nude mouse model prepared with the PANC-1 （K-ras＋） and SW1990 （K-ras-） cell lines. Results： Compared with K-ras＋peptide, low concentration K-ras＋pepUde-CNP can be effectively presented by DCs （P 〈 0.05）. CTLs induced by DCs pulsed with whole tumor antigen had significant greater killing effect （P 〈 0.05） on PANC-1 and SW1990 pancreatic cancer cells compared with K-ras＋peptide and K-ras＋peptide-CNP-induced CTLs. CTLs induced by DCs pulsed with K-ras＋peptide and K-ras＋peptide-CNP had a specific killing effect （P 〈 0.05） for PANC-1 and no effect （P 〉 0.05） on SW1990 cell lines （P 〉 0.05）. Conclusion： Cationic nanoparticles with K-ras （12-Val） mutant peptide can be effectively presented by DCs at a low concentration in a short time. CTLs induced by K-ras＋peptide-CNP had specific killing activity for the pancreatic cancer cell line with the K-ras （12-Val） mutant and could significantly inhibit tumor growth and increase the survival time of tumor-bearing nude mice.

Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

We have developed and tested chimeric T-cell receptors (TCR) specific for p185HER2. In these experiments, retroviral vectors expressing the N29γ or N29ζ receptors were constructed in pRET6. Amphotropic viral producer cells were established in the GALV-based PG13 packaging cell line. Ficoll purified human peripheral blood lymphocytes (PBL) were virally transduced using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28 monoclonal anti- bodies, followed by viral infection in the presence of fibronectin fragment CH296. Transduced cells were co-cultured with human tumor cell lines that overexpress (SK-OV-3) or underexpress (MCF7) p185HER2 to assay for antigen specific im- mune responses. Both CM+ and CD8+ T-cells transduced with the N29γ or N29ζ chTCR demonstrated HER2-specific anti- gen responses, as determined by release of Th1 like cytokines, and cellular cytotoxicity assays. Our results support the fea- sibility of adoptive immunotherapy with genetically modified T-cells expressing a chTCR specific for p185HER2.

A preliminary analysis of antineoplastic activity ofparvovirus MVMp NS-1 proteins

Human gastric cancer MKN-45 cells were transfectedwith pULB 3238, a plasmid carrying MVMp NS-1 genewith its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectantsdied, while others remained alive, but the growth featuresof survived cells were changed. For further study on theantineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established byconventional method. Among 4 founders, one of them wasfound to be able to transmit the transgene to around 50%of their offsprings. RT-PCR was performed to indicate theexpression of NS-1 gene in transgenic mice and its mRNAappeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

Anti-Tumor Efficacy of Gene Vaccine Expressing PSMA

OBJECTIVE To observe anti-tumor effects of PVAX-PSMA gene vaccine. METHODS The PSMA gene was inserted into a mammalian expression vector, PVAX-1, to construct the DNA vaccine candidate, and was then used to vaccinate C57BL/6 mice. Animals vaccinated with PVAX-1 and NaC1 were used as controls. Anti-PSMA antibody was detected in sera of the animals. The proliferation and cytotoxicity of the spleen cells were observed. The immunized mice were inoculated with RM-1 cells. The mice were inoculated with RM-1 cells, and then the mice were immunized. The anti-tumor efficacy of the gene vaccine was evaluated by the ratio of tumor formation, tumor volume, tumor mass before and after gene vaccination and evaluated by survival rate of the immunized mice. RESULTS High level of anti-PSMA antibody was induced in the PVAX-PSMA group. The splenocytes from PVAX-PSMA group were stimulated to produce strong proliferation responses and significant cytotoxic T-cells （CTL） activity. After the mice were immunized with PVAX-PSMA gene, tumor occurrence was decreased, and the growth velocity of tumor was markedly reduced, resulting in prolonged tumor-free time （P 〈 0.05）. CONCLUSION PVAX-PSMA gene vaccine has significant anti- tumor effects and provides an experimental basis for primary prevention and immunotherapy of prostate cancer.

The effect of Tagalsin on mice with transplanted H22 Hepatocarcinoma

Objective： The aim of our study was to explore the inhibitory effect of Tagalsin on murine transplanted tumour and its anti-tumour mechanisms. Methods： Animal models were established by transplanting H22 hepatoma cells to the left oxter of mice, and ten days later they were randomly divided into five groups： blank control group （edible oil）, positive control group （HCFU） and Tagalsin group, including low-dose, middle-dose and high-dose group. All mice were killed 24 h after medication, during which observation was conducted concerning survival conditions, body weight changes, spleen weight and tumor weight of tumor-bearing mice; the spleen index and the tumor inhibitor rate （IR） were calculated and pathological changes of tumor-bearing mice were observed by HE dye. Apoptosis factors p53 and Survivin mRNA were detected by reverse transcription polymerase chain reaction （RT-PCR）. Results： Tagalsin can inhibit hepatoma growth effectively without influencing spleen index and body weight, the tumor inhibitor rate （IR） of low, middle and high dose group of Tagalsin were 15.81%, 36.75% and 74.79% respectively, the tumor inhibitor rate （IR） of HCFU were 73.93%. Apoptosis cells could be found from the specimen of the positive control group and Tagalsin groups. Reverse transcription polymerase chain reaction （RT-PCR） results showed that positive control group’s and Tagalsin treatment groups’ p53 gene expression enhanced significantly and Survivin gene expression dropped comparing with blank group （P 0.05）. Conclusion： Tagalsin can inhibit growth of the H22 hepatoma cells significantly, the mechanism of anti-tumor effect may work by up-regulating p53 expression and down-regulating Survivin expression. Tagalsin may be considered as a potential candidate for chemoprevention.

Research on construction and identification of lentiviral vector of expressing miRNA targeting IGF1R gene regulated by survivin promoter and its inhibition to liver cancer cell growth

Objective： The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter. Methods： The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME to recombinant plasmid sur-pPRIME. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the sur-pPRIME vector, named sur-pPRIME-IGF1R-miR30-shRNA. Viruses were propagated on 293T cells. Viruses were purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The effect of sur-pPRIME-IGF1R-miR30-shRNA on IGF1R expression of Hep3B cells was detected by RT-PCR and Western blot. The antitumor potential of sur-pPRIME-IGF1R-miR30-shRNA to Hep3B cells was evaluated by CCK-8 assay. Results： sur-pPRIME-IGF1R-miR30-shRNA was constructed successfully. Functional PFU titers of sur-pPRIME-IGF1R-miR30-shRNA were 4.58×10^9 PFU/rnL. Sur-pPRIME-IGF1R-miR30-shRNA was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of Hep3B cells. Conclusion： sur-pPRIME-IGF1R-miR30-shRNA expressing IGF1R-siRNA can inhibit IGF1R expression and may be used for further investigation of gene therapy of liver cancer.