Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 （AdhIL-24） on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice （n=10 for each group） bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density （MVD）.Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0×10^10 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo （P 〈0.05）. The intratumoral MVD decreased significantly in the treated tumors （P 〈0.05）.Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

基于PI3K/Akt/mTOR信号通路的miR-24调控ox-LDL诱导的HUVECs自噬机制研究

目的探讨miR-24对氧化低密度脂蛋白(ox-LDL)诱导下的人脐静脉内皮细胞(HUVECs)自噬的影响及其相关机制,为进一步阐明miR-24在动脉粥样硬化(AS)中的作用提供理论依据。方法采用实时荧光定量PCR(qRT-PCR)检测miR-24的表达;应用蛋白免疫印迹法(western blot)和qRT-PCR法检测Beclin-1、LC3Ⅰ/LC3Ⅱ、p-mTOR、p-PI3K、p-Akt的蛋白和mRNA表达水平;应用透射电子显微镜技术检测细胞的自噬小体生成情况;应用四甲基偶氮唑盐(MTT)法、细胞划痕法、Caspase-3比色法和Hoechst 33258染色法分别检测细胞活性、迁移和凋亡情况。结果应用ox-LDL诱导HUVECs后,发现HUVECs中miR-24的表达显著降低(P<0.05)。miR-24过表达可明显抑制ox-LDL诱导的HUVECs自噬(P<0.05),而miR-24低表达则会增加ox-LDL诱导的HUVECs自噬(P<0.05)。miR-24过表达可显著降低Beclin-1的表达水平,上调LC3Ⅰ/LC3Ⅱ的水平(P<0.05),同时,miR-24过表达可显著促进p-PI3K、p-Akt和p-mTOR的表达(P<0.05)。此外,miR-24过表达显著抑制HUVECs的活力和迁移,增加Caspase-3活性并促进其凋亡(P<0.05)。结论miR-24的过表达可激活PI3K/Akt/mTOR信号通路而降低ox-LDL诱导的HUVECs的自噬水平并促进其凋亡,miR-24可能成为AS的潜在治疗新靶点。

Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells

AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin(IL)-24 in hepatocellular carcinoma(HCC) cells.METHODS: γ-secretase inhibitors(GSIs) were used to down-regulate Notch1.Hep G2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h.Cell viability was measured by MTT assay.The cellular and nuclear morphology was observed under a fluorescence microscope.To further verify the apoptotic phenotype,cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining.The expression of Notch1,SNAIL1,SNAIL2,E-cadherin,IL-24,XIAP and VEGF was detected by Western blot.The invasion and migration capacities of HCC cells were detected by wound healing assays.Notch1 and Snail were downregulated by RNA interference,and the target proteins were analyzed by Western blot.To investigate the mechanism of apoptosis,we analyzed Hep G2 cells treated with si Notch1 or si CON plus IL-24 or not for 48h by caspase-3/7 activity luminescent assay.RESULTS: GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in Hep G2 cells.The addition of 50 ng/m L IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%,respectively.Treatment with IL-24 alone did not induce any cytotoxic effect.In SMMC7721 cells with the addition of IL-24 to GSI-I(2.5 μmol/L),the reduction of cell viability was only about 25%.Following GSI-I/IL-24 combined treatment for 6 h,the apoptotic rate of Hep G2 cells was 47.2%,while no significant effect was observed in cells treated with the compounds employed separately.Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in Hep G2 cells.Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I.Furthermore,the increased GSI-I and IL-24 in Hep G2 cell was associated with downregulation of MMP-2,XIAP and VEGF.In the absence of treatment,Hep G2 cells could migrate into the scratched space in 24 h.With IL-24 or GSI-I treatment,the wound was still open after 24 h.And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I.Treatment of Notch-1 silenced Hep G2 cells with 50 ng/m L IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination.Caspase-3/7 activity was increased in the presence of si Notch1 plus IL-24 treatment.CONCLUSION: Down-regulation of Notch1 by GSI-I or si RNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of Hep G2 cells.

Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients

AIM To evaluate the treatment effects of recombinant human interleukin-12(rh IL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc.METHODS The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy(IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rh IL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervenedwith rh IL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS Rh IL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period.CONCLUSION Rh IL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

Interleukin-24 is correlated with differentiation and lymph node numbers in rectal cancer

AIM:To assess the significance of interleukin(IL)-24 and vascular endothelial growth factor(VEGF)expression in lymph-node-positive rectal cancer. METHODS:Between 1998 and 2005,90 rectal adenocarcinoma patients with lymph node involvement were enrolled.All patients received radical surgery and postoperative pelvic chemoradiotherapy of 50.4-54.0 Gy.Chemotherapy of 5-fluorouracil and leucovorin or levamisole was given intravenously during the first and last week of radiotherapy,and then monthly for about 6 mo.Expression of IL-24 and VEGF was evaluated by immunohistochemical staining of surgical specimens, and their relations with patient characteristics and survival were analyzed.The median follow-up of surviving patients was 73 mo(range:52-122 mo). RESULTS:IL-24 expression was found in 81 out of 90 patients;31 showed weak intensity and 50 showedstrong intensity.VEGF expression was found in 64 out of 90 patients.Negative and weak intensities of IL-24 expression were classified as negative expression for analysis.IL-24 expression was significantly reduced in poorly differentiated tumors in comparison with well or moderately differentiated tumors(P=0.004),N2b to earlier N stages(P=0.016),and stageⅢc to stageⅢ a orⅢb(P=0.028).The number of involved lymph nodes was also significantly reduced in IL-24-positive patients in comparison with IL-24-negative ones. There was no correlation between VEGF expression and patient characteristics.Expression of IL-24 and VEGF was not correlated with survival,but N stage and stages were significantly correlated with survival. CONCLUSION:IL-24 expression was significantly correlated with histological differentiation,and inversely correlated with the degree of lymph node involvement in stageⅢrectal cancer.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Recombinant human interleukin-11 for treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer

Objective： To evaluate the efficacy and safety of recombinant human interleukin-11 （rhIL-11） for the chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer. Methods： It was an opened and non-randomized controlled clinical study. When the platelet counts was under 75 × 10^9/L after chemotherapy, rhlL-11 was administered 25 μg/（kg·d） as a daily SC injection last for 7-14 days, or discontinued when platelet counts 〉 100 × 10^9/L. Results： Seventysix patients were enrolled into this study. The treatment group and the control group had thirty-eight cases, respectively. The mean recovery time to PLT ≥ 100 × 10^9/L was 8.1 days in treatment group, while in control group was 12.2 days （P 〈 0.01）. Moreover, the mean recovery time from PLT 〈_ 50 × 10^9/L to 〉 100 × 10^9/L was 8.9 days in treatment group, while in control group was 12.9 days （P 〈 0.05）. There was a statistical difference between the two groups. Major side effects included edema, fever, articular muscle soreness, but they were all mild and well tolerable. Conclusion： rhIL-11 can be safely and effectively used for the treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer.

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Artificial nerve graft constructed by coculture of activated Schwann cells and human hair keratin for repair of peripheral nerve defects

Studies have shown that human hair keratin(HHK) has no antigenicity and excellent mechanical properties. Schwann cells, as unique glial cells in the peripheral nervous system, can be induced by interleukin-1β to secrete nerve growth factor, which promotes neural regeneration. Therefore, HHK with Schwann cells may be a more effective approach to repair nerve defects than HHK without Schwann cells. In this study, we established an artificial nerve graft by loading an HHK skeleton with activated Schwann cells. We found that the longitudinal HHK microfilament structure provided adhesion medium, space and direction for Schwann cells, and promoted Schwann cell growth and nerve fiber regeneration. In addition, interleukin-1β not only activates Schwann cells, but also strengthens their activity and increases the expression of nerve growth factors. Activated Schwann cells activate macrophages, and activated macrophages secrete interleukin-1β, which maintains the activity of Schwann cells. Thus, a beneficial cycle forms and promotes nerve repair. Furthermore, our studies have found that the newly constructed artificial nerve graft promotes the improvements in nerve conduction function and motor function in rats with sciatic nerve injury, and increases the expression of nerve injury repair factors fibroblast growth factor 2 and human transforming growth factor B receptor 2. These findings suggest that this artificial nerve graft effectively repairs peripheral nerve injury.

Upregulation of stromal cell-derived factor-1 alpha/CXCR4 axis-induced migration of human neural progenitors by tumor necrosis factor-alpha and interleukin-8

BACKGROUND： Studies of several animal models of central nervous system diseases have shown that neural progenitor cells （NPCs） can migrate to injured tissues. Stromal cell-derived factor 1 alpha （SDF-la）, and its primary physiological receptor CXCR4, have been shown to contribute to this process. OBJECTIVE： To investigate migration efficacy of human NPCs toward a SDF-1α gradient, and the regulatory roles of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） in SDF-1α/CXCR4 axis-induced migration of NPCs. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, cellular and molecular biology study was performed at the Laboratory of Department of Cell Biology, Medical College of Soochow University between October 2005 and November 2007. MATERIALS： SDF-1α and mouse anti-human CXCR4 fusion antibody were purchased from R＆D Systems, USA. TNF-αwas purchased from Biomyx Technology, USA and IL-8 was kindly provided by the Biotechnology Research Institute of Soochow University. METHODS： NPCs isolated from forebrain tissue of 9 to 10-week-old human fetuses were cultured in vitro. The cells were incubated with 0, 20, and 40 ng/mL TNF-α, or 0, 20, and 40 ng/mL IL-8, for 48 hours prior to migration assay. For antibody-blocking experiments, cells were further pretreated with 0, 20, and 40 μg/mL mouse anti-human CXCR4 fusion antibody for 2 hours. Subsequently, the transwell assay and CXCR4 blockade experiments were performed to evaluate migration of human NPCs toward a SDF-1α gradient. Serum-free culture medium without SDF-1α served as the negative control. MAIN OUTCOME MEASURES： The transwell assay was performed to evaluate migration of human NPCs toward a SDF-1α gradient, which was blocked by fusion antibody against CXCR4. In addition, CXCR4 expression in human NPCs stimulated by TNF-α and IL-8 was measured by flow cytometry. RESULTS： Results from the transwell assay demonstrated that SDF-1α was a strong chemoattractant for human NPCs （P 〈 0.01）, and 20 ng/mL produced the highest levels of migration. Anti-human CXCR4 fusion antibody significantly blocked the chemotactic effect （P 〈 0.05）. Flow cytometry results showed that treatment with TNF-α and IL-8 resulted in increased CXCR4 expression and greater chemotaxis efficiency of NPCs towards SDF-1α（P 〈 0.01）. CONCLUSION： These results demonstrated that SDF-la significantly attracted NPCs in vitro, and neutralizing anti-CXCR4 antibody could block part of this chemotactic function. TNF-α and IL-8 increased chemotaxis efficiency of NPCs towards the SDF-1αgradient by upregulating CXCR4 expression in NPCs.

RESPONSES OF HUMAN FETAL SPLENOCYTES AND THYMOCYTES TO INTERLEUKIN-2: LAK ACTIVITY AND PROLIFERATION

Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.

Mechanism of piR-1245/PIWI-like protein-2 regulating Janus kinase-2/signal transducer and activator of transcription-3/vascular endothelial growth factor signaling pathway in retinal neovascularization

Inhibiting retinal neovascularization is the optimal strategy for the treatment of retina-related diseases, but there is currently no effective treatment for retinal neovascularization. P-element-induced wimpy testis(PIWI)-interacting RNA(piRNA) is a type of small non-coding RNA implicated in a variety of diseases. In this study, we found that the expression of piR-1245 and the interacting protein PIWIL2 were remarkably increased in human retinal endothelial cells cultured in a hypoxic environment, and cell apoptosis, migration, tube formation and proliferation were remarkably enhanced in these cells. Knocking down piR-1245 inhibited the above phenomena. After intervention by a p-JAK2 activator, piR-1245 decreased the expression of hypoxia inducible factor-1α and vascular endothelial growth factor through the JAK2/STAT3 pathway. For in vivo analysis, 7-day-old newborn mice were raised in 75 ± 2% hyperoxia for 5 days and then piR-1245 in the retina was knocked down. In these mice, the number of newly formed vessels in the retina was decreased, the expressions of inflammationrelated proteins were reduced, the number of apoptotic cells in the retina was decreased, the JAK2/STAT3 pathway was inhibited, and the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor were decreased. Injection of the JAK2 inhibitor JAK2/TYK2-IN-1 into the vitreous cavity inhibited retinal neovascularization in mice and reduced expression of hypoxia inducible factor-1α and vascular endothelial growth factor. These findings suggest that piR-1245 activates the JAK2/STAT3 pathway, regulates the expression of hypoxia inducible factor-1α and vascular endothelial growth factor, and promotes retinal neovascularization. Therefore, piR-1245 may be a new therapeutic target for retinal neovascularization.

重组hIL-24原核表达载体的构建及表达

采用基因克隆的方法构建人白细胞介素(hIL-24)基因的原核表达载体pET-28a(+)-hIL24,在大肠杆菌中诱导表达重组蛋白rhIL-24,SDS-PAGE和Western blotting分析蛋白表达,探讨其最佳诱导表达条件。结果表明:hIL-24基因的表达产物分子质量约25 ku,且以包涵体的形式存在于超声裂解后的沉淀中,重组蛋白rhIL-24可特异性被鼠抗人IL-24单克隆抗体识别。当IPTG终浓度为0.5 mmol.L-1、37℃诱导8 h时,其表达量最高。hIL-24基因在该原核表达系统中的成功表达,为其生物学功能的研究奠定了基础。

人白介素24基因诱导人宫颈癌Ca Ski细胞凋亡的实验研究

目的:研究人白介素24(hIL-24)在体外诱导宫颈癌CaSki细胞凋亡及其作用机制.方法:①以脂质体包裹重组真核表达质粒pcDNA3.1(+)-hIL-24转染Ca Ski细胞;②利用RT-PCR技术检测处理后Ca Ski细胞中HPV E6癌基因的mRNA水平变化;③Western Blot分析处理后Ca Ski细胞中抑癌蛋白P53水平的变化;④流式细胞仪分析处理后的Ca Ski细胞凋亡情况.结果:处理后Ca Ski细胞中HPV E6癌基因的mRNA水平下降,抑癌蛋白P53水平增加,细胞凋亡率升高.结论:真核表达载体介导的hIL-24基因体外转染宫颈癌Ca Ski细胞后,能抑制Ca Ski细胞中HPV E6癌基因的表达,使抑癌蛋白P53恢复活性,促使宫颈癌Ca Ski细胞凋亡.

腺病毒介导的白细胞介素24基因对人肺腺癌细胞的放疗增敏作用

目的研究腺病毒介导的白细胞介素24基因(Ad-IL-24)对人肺腺癌细胞SPC-A1体外抑癌效应、放疗增敏作用。方法将Ad-IL-24感染的SPC-A1细胞,用逆转录-聚合酶链反应(RT-PCR)及蛋白印迹(Western blot)法检测IL-24目的基因在SPC-A1细胞中的转录和表达;四甲基偶氮唑蓝(MTT)法检测磷酸盐缓冲液(PBS)组、腺病毒(Ad-GFP)组、Ad-IL-24组、放疗组、Ad-GFP联合放疗组(Ad+放疗组)及Ad-IL-24联合放疗组(Ad-IL-24+放疗组)对SPC-A1细胞生长抑制作用,流式细胞术(FCM)检测各组SPC-A1细胞生长周期和凋亡率。RT-PCR法检测SPCA1细胞中生存素、半胱氨酸蛋白酶3(Caspase-3)、B细胞淋巴瘤/白血病基因伴随蛋白X(bax)、B细胞淋巴瘤/白血病基因-2(bcl-2)凋亡相关因子的表达。结果 Ad-IL-24作用SPC-A1细胞后,目的基因在SPC-A1细胞中成功转录及表达;Ad-IL-24能显著抑制SPC-A1细胞的生长且呈现时间依赖性,Ad-IL-24组(31.1±0.9)%、放疗组(44.4±2.3)%、Ad-IL-24+放疗组(72.4±1.5)%的生长抑制率分别与Ad-GFP组(2.7±1.1)%比较,均差异有统计学意义(P<0.01),且Ad-IL-24+放疗组优于Ad-IL-24组、放疗组(F=314.613,P<0.01),呈现放疗增敏的协同作用(Q=1.172);Ad-IL-24可诱导SPC-A1细胞G2/M期阻滞和细胞凋亡,Ad-IL-24+放疗组(40.6±3.3)%的细胞凋亡率与Ad-IL-24组(15.4±1.1)%、放疗组(26.3±1.7)%比较,差异有统计学意义(F=87.194,P<0.01),且具有放疗增敏协同作用(Q=1.162)。Ad-IL-24能明显上调促细胞凋亡相关因子bax、Caspase-3,并下调细胞抗凋亡因子bcl-2、生存素的表达,Ad-IL-24+放疗组作用优于Ad-IL-24组、放疗组。结论 Ad-IL-24有放疗增敏的作用,是理想的放疗增敏剂,该作用机制可能与诱导肿瘤细胞阻滞在G2/M期以及促进肿瘤细胞凋亡有关。

人泡沫病毒介导IL-24对肿瘤细胞的抑制作用

目的:利用重新构建的人逆转录病毒四载体系统共转染人胚肾细胞获得携带IL-24的复制缺陷型重组人泡沫病毒(HFV-IL24)颗粒,探讨复制缺陷型人泡沫病毒介导IL-24对肿瘤细胞生长分化的抑制作用。方法:重组构建含IL-24的人泡沫病毒载体质粒(pΔΦ-IL24);与辅助质粒共转染人胚肾HEK293T细胞后获取重组病毒颗粒(HFV-IL24);RT-PCR检测目的基因的表达;MTT法检测人宫颈癌HeLa细胞的体外增殖水平,台盼蓝染色计数检测细胞存活率,流式细胞术检测细胞活性和周期变化。结果:成功构建含IL-24的人泡沫病毒载体(pΔΦ-IL24);收获得到复制缺陷型重组人泡沫病毒颗粒(HFV-IL24);感染后的HeLa细胞显示出明显的生长抑制现象(P<0.01)并呈现周期变化和较高的凋亡(死亡)率。结论:所构建的重组病毒载体pΔΦ-IL24及由此而产生的重组人泡沫病毒颗粒(HFV-IL24)的功能性检测加深对HFV和抗癌基因IL-24的了解,为泡沫病毒载体的应用和IL-24抗肿瘤的基因治疗提供了研究方向和理论依据。

人IL-24基因在CHO细胞中的表达及其抗肿瘤效应

目的构建人IL-24基因真核表达载体,在CHO细胞中进行稳定表达,并检测重组表达蛋白rhIL-24的抗肿瘤活性。方法将测序验证的人IL-24基因亚克隆至真核表达载体pcDNA3,构建重组真核表达载体pcDNA3-hIL-24,转染CHO细胞进行稳定表达,经RT-PCR鉴定后用MTT法、Ho-echst染色和流式细胞术检测CHO细胞表达的rhIL-24诱导A549人肺腺癌细胞凋亡的抗肿瘤效应,用ELISA检测其刺激免疫细胞分泌IL-6和IFN-γ的功能。结果经双酶切和PCR鉴定,重组真核表达载体构建正确,人IL-24在CHO细胞中获得稳定表达,且所表达的人IL-24具有较强的诱导A549人肺腺癌细胞凋亡及上调免疫细胞表达IL-6和IFN-γ的免疫刺激活性。结论人IL-24基因的稳定表达和抗肿瘤效应的实验研究,为进一步研究人IL-24抗肿瘤的分子机制及潜在的应用奠定了基础。

人IL-24基因的克隆及在COS-7细胞中的表达

目的 :克隆人IL 2 4基因 ,构建真核表达载体 ,在COS 7细胞中进行表达 ,并检测重组表达蛋白hIL 2 4的抗肿瘤活性。方法 :分离人外周血单个核细胞 ,提取细胞总RNA ,采用RT PCR技术克隆人IL 2 4基因 ,将其重组于pUC19,双脱氧末端终止法测定其核苷酸序列 ,构建真核重组表达载体pcDNA3 hIL 2 4 ,进行双酶切和PCR鉴定 ,以质粒pcDNA3 hIL 2 4转染COS 7细胞进行瞬时表达 ,用RT PCR检测mRNA表达 ,并用MTT法、TUNEL法和流式细胞术检测其诱导肿瘤细胞凋亡的活性。结果 :成功获得 6 2 1bp的人IL 2 4基因 ,测序正确 ,经双酶切和PCR鉴定真核表达质粒构建正确 ,以脂质体转染COS 7细胞后 ,用RT PCR法、MTT法、TUNEL法和流式细胞术检测表明COS 7细胞可表达hIL 2 4 ,且所表达的人IL 2 4具有较强的诱导A5 4 9肺腺癌细胞凋亡的作用。结论 :人IL 2 4基因的瞬时表达和凋亡效应的初步研究已获成功 ,为研究人IL 2