核心蛋白聚糖协同白细胞介素24对人外周血单个核细胞作用的体外研究

Objective To investigate the co-effect of decorin (DCN) and interleukin-24 (IL-24) on proliferation and Interferon-γ (IFN-γ) secretion of human peripheral blood mononuclear cells (PBMCs). Methods Recombinant plasmid pcDNA3. 1 (+)-DCN and pcDNA3. 1 (+)-IL-24 were constructed and transfected into PBMCs by liposome. After transfected with different plasmids, the PBMCs were divided into 6 groups;blank control group, Lipofectamine TM group, empty vector group, DCN group, IL-24 group, DCN and IL-24 group. PBMC proliferation was determined by methyl thiazolyl tetrazolium assay. IFN-7 expression in the supernatant was detected by ELISA. Flow cytometry was used to determine the surface expression of programmed death-1 (PD-1) on PBMCs. Statistical analysis was made using LSD-t test. Results The recombinant plasmid pcDNA3. 1 (+)-IL-24 was constructed successfully. PBMC proliferation and IFN-7 secretion were significantly higher in DCN and IL-24 group, and the expression of PD-1 was also upregulated obviously. Conclusion The combination of DCN and IL-24 could promote PBMC proliferation and strengthen the function of PBMCs in vitro.     

核心蛋白聚糖协同白细胞介素24对人外周血单个核细胞作用的体外研究

目的 探讨核心蛋白聚糖(decorin,DCN)联合白细胞介素24(interleukin-24,IL-24)对人外周血单个核细胞(human peripheral blood mononuclear cell,PBMC)增殖及分泌干扰素γ(Interferon-γ,IFN-γ)的影响.方法 构建重组质粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24,并利用脂质体将两种质粒转染PBMC.根据转染质粒的不同将PBMC分为空白对照组、脂质体组、空质粒组、DCN组、IL-24组、DCN与IL-24联合组.采用四甲基偶氮唑蓝比色法检测PBMC的增殖,ELISA测定细胞培养上清中IFN-γ的表达,流式细胞技术检测PBMC表面程序性死亡分子1(PD-1)的表达.采用LSD-t检验进行统计学分析.结果 重组质粒pcDNA3.1(+)-IL-24构建成功.与其他组相比,DCN与IL-24联合能显著提高PBMC增殖率和IFN-γ分泌量,同时也能上调PD-1的表达水平.结论 DCN与IL-24双基因联合在体外能促进PBMC的增殖,并能增强PBMC的功能.     

Overexpression of acetylcholinesterase inhibited cell proliferation and promoted apoptosis in NRK cells

AIM: To study the potential function of acetylcholinesterase (AChE) in apoptosis through overexpression of AChE in Normal Rat Kidney (NRK) cells. METHODS: AChE activity was detected by the method of Karnovsky and Roots. Activated caspase-3 was analyzed by Western blotting and immunoflurescence with antibody special to activated caspase-3 fragment. The expression plasmids were constructed in pcDNA3.1 containing AChE gene or a fragment of AChE antisense that were got from RT-PCR. Stable expression cell lines were selected by G418 in cells transfected by lipofection. AChE expression was analyzed by RT-PCR and Western blotting. The proliferation rates of transfected cells were examined by the growth curve and cloning efficiency. MTT assay was used to analyze the cell viability. RESULTS: The proliferation rate of the cells transfected with AChE was retarded and the cloning efficiency was lower (28.2 %±3.1 % and 48.7 %±2.1 %) than cells transfected with vector (56.1 %±0.3 %) or AChE-antisense (77.7 %±2.2 %     

ntranasal co-delivery of IL-6 gene enhances the mmunogenicity of anti-caries DNA vaccine

Aim： To investigate the effects of co-delivering 11_-6 expressing plasmid pCI-IL-6 on the immunogenicity of the anti-caries DNA vaccine pClA-P, which encodes the surface protein antigen PAc of Streptococcus mutans. Methods： Plasmid pCI-IL-6 was constructed by inserting the murine IL-6 gene into the pCI vector. Expression of IL-6 in vitro was assessed using Western blot analysis. BALB/c mice were intranasally co-immunized with pClA-P plus pCI-IL-6 on d 0 and 14. Anti-PAc IgG and secretory IgA （slgA） were assessed by ELISA. Splenocytes from the mice were re-stimulated with the PAc protein, and IFN-y and IL-4 production was measured using ELISA. Splenocyte proliferation was analyzed with flow cytometry. Rats were similarly immunized, and dental caries scores were determined using the Keyes method. Results： Marked expression of IL-6 was found in C0S-7 cells transfected with pCI-IL-6. In the pCI-IL-6 co-immunized mice, the specific IgG antibodies in serum and slgA antibodies in saliva were significantly higher than those in the control mice at weeks 4 and 8. Moreover, the secretion of IFN-y from splenocytes in response to re-stimulation with PAc protein was significantly higher in the pCI-IL-6 co-immunized mice than that in the control mice, whereas the secretion of IL-4 had no significant difference. The proliferation of splenocytes from the pCI-IL-6 co-immunized mice was significantly higher than that from the mice immunized with pClA-P and pCl vector. In the rat caries model, the pCI-IL-6 co-immunization rats displayed lower caries scores than the control rats. Conclusion： Intranasal co-delivery of IL-6 gene significantly enhances the immunogenicity of the anti-caries DNA vaccine.     

The effects of overexpression of Nurrl, a midbrain-specific transcription factor, on SK -N -SH cells vulnerability to neurotoxin 6-OHDA

AIM The aim of this study is to investigate the possible correlation between the expression of Nurrl gene and selective cell death of dopamine (DA) neurons. Nurrl ( nuclear receptor-related factor 1 ) is highly expressed in mesencephalic DA system and specifically required for development and survival of DArgic neurons, the cells primarily lost and aggressive degenerated in Parkinson’s disease (PD). METHODS Firstly, SK-N-SH cells, which do not express endogenous Nurrl, were transfected with the pBK-RSV-Nurrl plasmid by LipofectAMINE and selected by G418. The expression of Nurrl protein in SK-N-SH/Nurrl cells was determined by immunocytochemistry. The effect of Nurrl gene on cell proliferation was also investigated.     

Notch1 signaling inhibits growth of EC109 esophageal carcinoma cells through downmodulation of HPV18 E6/E7 gene expression

Aim： To investigate the role of the Notch1 signaling pathway in growth arrest of an esophageal carcinoma cell line （EC109） in vitro and the mechanism involved. Methods： An intracellular domain of Notch1 （ICN） was transfected into cultured EC109 cells by lipofectamine transfection. Subsequently, the proliferation of the transfected cells was measured by an MTT assay. Cell cycle distribution was analyzed by flow cytometry. Human papillomavirus type 18 （HPV18） E6/E7 mRNA expression was detected by RT-PCR, and p53 protein expression was detected by Western blot. Results： Activation of Notch1 signaling resulted in inhibition of EC109 cell proliferation with the induction of G2/M arrest, downmodulation of HPV18 E6/E7 gene expression, and upregulation of p53 expression. Conclusion：Repression of HPV18 E6/E7 expression by Notch1 signaling results in the activation of p53-mediated pathways with concomitant growth suppression of HPV18-positive EC109 cells.     

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells

Aim： Somatostatin receptor subtype 2 （SSTR2） is the principal mediator of somatostatin＇s （SST） antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved.
Methods： SSTR2 expression levels were determined by qRToPCR in several tumor cell lines. Then, a plasmid plRES2-EGFP-SSTR2 （pSlG） was constructed and stably transfected into MCF-7 cells （MCF-7/pSIG）. After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor （EGFR） expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT.
Results： The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G1/S cell cycle arrest. Furthermore the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSlG cells.
Conclusion： Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G1/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.     

Overexpression of coxsackie and adenovirus receptor inhibit growth of human bladder cancer cell in vitro and in vivo

Aim： To study the effect of the overexpression of coxsackie and the adenovirus receptor （CAR） on the growth of the human bladder cancer cell in vitro and in vivo. Methods： A retroviral vector pLXSN-CAR expressing CAR was constructed and confirmed by restriction enzyme mapping. The pLXSN-CAR vector and con- trol vector pLXSN were transfected into the PT67 packaging cell line to generate retrovirus with high titer. The CAR-negative T24 cell was infected with the pLXSNCAR and the pLXSN retrovirus, respectively. The positive clone cells were selected with G418 for 2 weeks. The expression level of the CAR protein was detected by Western blot assay. T24 cell growth in vitro was determined by 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Anchorage-independent growth was measured by soft-agar colony formation assay. In vivo cell growth was determined by a nude mice xenograft model. Results： The pLXSN-CAR vector containing full-length CAR cDNA was successfully constructed. Western blot analysis showed that a 46 kDa specific band was found in pLXSN-CA-transfected T24 cells. MTT assay identified the growth inhibition of T24/pLXSN-CAR cells. The cell colony forming ability of T24/pLXSNCAR cells was significantly lower than that of T24/pLXSN and parental T24 cells. There was a reduction in the tumor size in the T24/pLXSN-CAR group as compared with that of the T24/pLXSN group and parental T24 group. Conclusion： The overexpression of CAR in T24 bladder cancer cells can inhibit cell growth both in vitro and in vivo.