靶向STAT3的decoy核酸抑制膀胱癌细胞生长的实验研究

目的观察特异性靶向STAT3的decoy寡核苷酸（0DNs）对膀胱癌细胞增殖和凋亡的影响，并探讨其作用机理。方法通过体外细胞培养技术，将STAT3 decoy ODNs转染入膀胱癌T24细胞内，台盼蓝排斥试验检测细胞活力，MTT法检测细胞增殖状态，Hoechst33258／PI荧光双染检测细胞凋亡特征，流式细胞仪检测细胞周期和早期凋亡，凝胶阻滞电泳检测STAT3的DNA结合活性，RT-P（、R检测STAT3下游靶基因Cyclin D1和Bcl—xL的mRNA表达水平。结果STAT3 decoy ODNs使膀胱癌细胞生长速度减慢，细胞增殖抑制率达75．0％；使细胞周期阻滞，S期细胞比率明显减少，由29．83％下降至1626％，而G0～G1期细胞比率明显增多，由50．82％上升至71．20％；促进膀胱癌细胞凋亡，细胞早期凋亡率高达50．75％；使STAT3的DNA结合活性下降，并使Cyclin D1、Bcl—xL的mRNA表达水平明显下降。结论STAT3 decoy ODNs可特异性靶向作用于STAT3蛋白，阻断STAT3的过度激活效应，通过下调Cyclin D1和Bel—xL的表达抑制膀胱癌细胞增殖，促进其凋亡。

Increased expressions of vascular endothelial growth factor(VEGF),VEGF-C and VEGF receptor-3 in prostate cancer tissue are associated with tumor progression

Aim： To investigate the differences in microvessel densities （MVD） and the expressions of vascular endothelial growth factor （VEGF）, VEGF-C and VEGF receptor-3 （VEGFR-3） between prostate cancer （PCa） tissues and adjacent benign tissues, and to explore the correlations among MVD, Jewett-Whitmore staging, Gleason scores and expressions of VEGF, VEGF-C and VEGFR-3 in the progression of PCa. Methods： An immunohistochemical approach was adopted to detect the expressions of CD34, VEGF, VEGF-C and VEGFR-3 in both cancer areas and peripheral benign areas of 71 primary prostatic adenocarcinoma specimens. A statistic analysis was then performed according to the experimental and clinic data. Results： Significantly upregulated expressions of VEGF, VEGF-C and VEGFR-3 were all found in malignant epithelium/cancer cells compared with adjacent benign epithelium （P 〈 0.01）. Patients in stage D had a significantly higher score than patients in stage A, B or C when comparing the expression of VEGF-C or VEGFR-3 in the tumor area （P 〈 0.01）. In addition, significant correlations were observed between Jewett-Whitmore staging and VEGF-C （rs = 0.738, P 〈 0.01）, clinical staging and VEGFR-3 （rs = 0.410, P 〈 0.01）, VEGF-C and Gleason scores （rs = 0.401, P 〈 0.01）, VEGFR-3 and Gleason scores （rs = 0.581, P 〈 0.001） and MVD and VEGF （rs = 0.492, P 〈 0.001）. Conclusion： Increased expressions of VEGF and VEGF-C were closely associ- ated with progression of PCa. The main contribution of increased VEGF expression for PCa progression was to upregulate MVD, which maintained the growth advantage of tumor tissue. However, the chief role of increased expressions of VEGF-C and VEGFR-3 was to enhance lymphangiogenesis and provide a main pathway for cancer cells to disseminate. （Asian J Androl 2006 Mar; 8： 169-175）

Expression of Vascular Endothelial Growth Factor-C and Vascular Endothelial Growth Factor Receptor-3 in Ovarian Epithelial Tumors

Objective： To explore the role of vascular endothelial growth factor-C （VEGF-C） in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. Methods： In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign tumors. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density （MVD） were assessed by image analysis. Results： VEGF-C mRNA and protein expression were detected in cytoplasm of carcinoma cells. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than those in borderline tumors and benign tumors （P〈0.05 or P〈0.01）. In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage Ⅲ-Ⅳ and with lymph node metastasis than those of clinical stage Ⅰ-Ⅱ and without lymph node metastasis respectively （P〈0.05 or P〈0.01）. VEGFR-3 positive vessels and MVD were significantly higher in VEGF-C protein positive tumors than negative tumors （P〈0.05）. VEGFR-3 positive vessels was significantly correlated with MVD（P〈0.01）. Conclusion： VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian tumors, and VBEGF-C could be used as a biologic marker of metastasis in ovarian epithelial tumors.

Study on the Expression of Dopamine Receptor-3(DRD3) in Rats after Sacral Spinal Cord Transection

[Objectives] This study aimed to study the distribution characteristics of DRD3(dopamine receptor-3) and the changes in its expression before and after spinal cord injury(SCI), in order to lay a morphological basis for later research. [Methods] Adult male Wistar rats were randomly divided into sham operation group and SCI group. The rat spinal cord transection model at the sacral 2(S_2) segment was established. Rat tail spasticity score was performed 60 d after SCI, and the rats with 4-5 points were screened for perfusion. The expression of DRD3 in the sacral spinal cord(S+C segment) was detected by immunofluorescence. [Results] In normal rats, DRD3 was mainly distributed in the dorsal horn(DH), intermediate zone(IMZ) and ventral horn(VH) of the gray matter. It was also expressed in the white matter of the spinal cord. After SCI, the distribution of DRD3 in the segment below the injury section was similar to that of normal rats. However, the expression was different(P<0.05). [Conclusions] There was no significant change in the distribution of DRD3 in spinal cord after SCI. After the spinal cord S_2 was completed transected, the expression of DRD3 was significantly reduced in the DH, IMZ and VH regions of the gray matter of the spinal cord.

Decoy receptor 3: Its role as biomarker for chronic inflammatory diseases

Members of the tumor-necrosis factor-α(TNF-α) and TNF-α receptor(TNFR) superfamilies of proteins(TNFSF and TNFRSF, respectively) play important roles in the function of the immune system. Decoy receptor 3(Dc R3, TNFRSF6b) is a decoy receptor that binds to three TNFSF ligands, Fas L, LIGHT and TL1 A. Association to these ligands competes with the corresponding functional receptors and blocks downstream signaling, leading to immunomodulatory effects, including the prevention of apoptosis. Dc R3 lacks a transmembrane region and exists only as a secreted protein, which is detectable in biological fluids. Recent studies have shown that Dc R3 is upregulated and may be pathogenetically implicated in several and diverse chronic inflammatory diseases. The strongest associations have been described for rheumatological diseases, mainly systemic lupus erythematosus and rheumatoid arthritis, inflammatory bowel disease, and serious infectious conditions, including systemic inflammatory response syndrome. In the majority of these conditions, Dc R3 m RNA and protein expression is elevated both at the target tissues as well as in the systemic circulation. Dc R3 concentration in the serum is untraceable in the majority of healthy individuals but can be detected in patients with various inflammatory diseases. In mostsuch cases, soluble Dc R3 correlates with disease severity, as patients with severe forms of disease have significantly higher levels than patients with milder or no activity. In addition, effective anti-inflammatory treatment leads to the disappearance of soluble Dc R3 from the circulation. Taken together, current evidence suggests that serum Dc R3 may become a useful biomarker for chronic inflammatory disorders, as it is upregulated in response to inflammatory stimuli, and may serve both as a prognostic marker for disease severity and as a surrogate indicator of response to treatment.

靶向阻断STAT3增强肝癌细胞H22对化疗药物阿霉素的敏感性

目的:探讨STAT3阻断剂Decoy ODN与临床常用肝癌化疗药阿霉素(Doxorubicin)、5-氟尿嘧啶(5-Fu)和顺铂(cisplatin)联合使用对肝癌的治疗效果及其对免疫系统的影响。方法:肝癌细胞转染Decoy ODN后用化疗药处理,以MTT法检测肝癌细胞的增殖能力,Annexin-V/7AAD双染法检测细胞凋亡率;对H22荷瘤小鼠进行Decoy ODN和阿霉素联合治疗,观察小鼠肿瘤生长情况以及生存期;流式细胞术检测治疗后小鼠PBMC的细胞分群和活化水平,以及阿霉素或阿霉素处理的肿瘤细胞对小鼠脾脏淋巴细胞体外活化作用。结果:转染Decoy ODN以后,阿霉素对H22细胞的抑制作用显著增强,H22细胞凋亡率也明显升高。Decoy ODN和阿霉素联合治疗可明显降低小鼠肿瘤生长速度并延长荷瘤小鼠的生存期;低剂量阿霉素增加了PBMC中T细胞的比例和CD69分子的表达,以及NK细胞CD107a和IFN-γ的表达;阿霉素处理的H22细胞可促进T细胞比例的升高。结论:Decoy ODN阻断肝癌细胞STAT3后可以增强肝癌细胞H22对阿霉素的敏感性,提高化疗效果,降低化疗毒副作用,改善机体免疫功能。

Relationship between LYVE-1, VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer

AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their lev- els and clinicopathological parameters in gastric cancer.METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic fea- tures such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the dis- tribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.

Pioglitazone ameliorates retinal ischemia/reperfusion injury via suppressing NLRP3 inflammasome activities

AIM：To explore the role of Pioglitazone（Pio） on a mouse model of retinal ischemia/reperfusion（I/R） injury and to elucidate the potential mechanism.METHODS：Retinal ischemia was induced in mice by increasing the intraocular pressure,and Pio was administered 4 h though periocular injection before I/R.The number of cells in the ganglion cell layer（GCL） was counted 7 d after retinal I/R injury.Glial fibrillary acidic protein（GFAP）,nuclear factor-kappa B（NF-κB）,p38,phosphorylated-p38,PPAR-γ,interleukin-1β（IL-1β）,Toll-like receptor 4（TLR4）,NLRP3,cleaved caspase-1,caspase-1 were determined by real-time polymerase chain reaction and Western blotting.RESULTS：Pio promoted the survival of retinal cells in GCL following retinal I/R injury（P〈0.05）.Besides,retinal I/R injury stimulated the expression of GFAP and TLR4,which were partially reversed by Pio treatment（P0.05）.Retinal I/R injury-upregulated expression of NLRP3,cleaved caspase-1,IL-1β was attenuated after Pio treatment（P〈0.05）.Moreover,I/R injury induced activation of NF-κB and p38 were inhibited by Pio treatment（P〈0.05）.CONCLUSION：Pio promotes retinal ganglion cells survival by suppressing I/R-induced activation of TLR4/NLRP3 inflammasomes via inhibiting NF-κB and p38 phosphorylation.

Serum VEGFR-3 and survival of advanced gastric cancer patients treated with FOLFOX

AIM: To explore if vascular endothelial growth factor receptor-3 (VEGFR-3) and carcinoembryonic antigen (CEA) can predict overall survival in advanced gastric cancer.METHODS: VEGFR-3 level was assessed by enzymelinked immunosorbent assay,and CEA was assessed by chemiluminescence immunoassay in the sera of 81 advanced gastric cancer patients before treatment with oxaliplatin plus 5-fluorouracil and folinic acid.RESULTS: Median survival time in patients with a low serum VEGFR-3 level was significantly longer than in those with a higher VEGFR-3 level (15.4 mo vs 7.7 mo,P < 0.001).Patients with a low CEA level had a longer survival than those with a higher CEA level (15.8 mo vs 8.6 mo,P < 0.001).Thirty-nine patients with low VEGFR-3 and low CEA levels had a median survival of 19.7 mo (P = 0.0006).The hazard ratio for patients with a high VEGFR-3 level was 2.443 (P = 0.002).CONCLUSION: High serum VEGFR-3 level is correlated significantly with poor survival.In patients with a high serum level of VEGFR-3,alternative chemotherapy regimens should be considered.

Expressions of Connexin and Par-3 in the Distal Margin of Rectal Cancer after Ultra-low Anterior Resection

This study examined the expression of connexin and protease-activated receptor 3 （par-3） in the distal resection margin of rectal cancer and the correlation of the expression of the two proteins with tumor relapse. A total of 40 patients with rectal cancer underwent ultra-low anterior resection with curved cutter stapler. The pathological specimens were divided into 3 groups in terms of sampling sites： tumor group, 2.0-cm group （in which the tissues were harvested 2.0 cm distal to the tumor tissues）, 3.0-cm group （in which the tissues were taken 3.0 cm away from the tumor tissues）. All the samples were pathologically observed and then measured for the expression of connexin and par-3 by employing immunohistochemistry and Western blotting. The operations in this series went uneventfully. No anastomotic stoma bleeding, stenosis and death occurred postoperatively. Histopathologically, in the tumor group, epithelial cells lost normal pattern of arrangement and polarity, and were loosely connected and even detached. In the 3.0-cm group, the epithelia had normal appearance, obvious cell polarity and essentially intact cell junction. Immunohistochemistry and Western blotting indicated that the 3.0-cm group had the strongest expression of connexin and par-3, and the expression in the 2.0-cm group and the tumor group was relatively weak. There existed significant difference in the expression of the two proteins among the three groups （P〈0.05 for all）. It was concluded that the down-regulated connexin and par-3 in the distal margin of rectal cancer tissues may indicate the progression of the disease and high likelihood of recurrence and metastasis. Although no tumor cells were found in the sections of the 2.0cm group, the decreased expression of connexin and par-3 may suggest the development of anaplasia and the increased odds of tumor relapse. Therefore, we are led to speculate that tumor resection only including 2.0 cm of unaffected rectum could not completely avoid the distant metastasis and local relapse.

靶向STAT3 decoy寡核苷酸对SLE患者PBMC中IFI16表达的影响及其意义

目的观察特异性靶向信号转导和转录激活子3(STAT3)的decoy寡核苷酸(ODNs)对系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)中干扰素诱导核蛋白16(IFI16)表达水平的影响。方法通过体外细胞培养术,将STAT3 decoy ODNs转染入提取的PBMC内,转染前后分别用反转录聚合酶链反应(RT-PCR)检测PBMC中IFI16的表达水平。结果转染STAT3 decoy ODNs前,IFll6 mRNA的表达水平在活动期、非活动期组与对照组比较明显增高,差异均有统计学意义(P＜0．05)。而活动期与非活动期组比较,差异无统计学意义(P＞0．05)。转染后,IFI16 mRNA的表达水平在活动期与非活动期组中均明显下降,与阴性对照比较差异均有统计学意义(P＜0．05),而正常组转染后与阴性对照比较差异无统计学意义(P＞0．05)。结论IFl16在SLE患者的异常高表达提示IFI16参与SLE的发病。SLE患者异常高表达的IFI16基因能被STAT3 decoy ODNs高度抑制,而在正常人不能被明显抑制,提示SLE患者异常高表达的IFI16是受JAK-STAT3信号转导途径调控的。

诱骗寡聚脱氧核苷酸在疾病治疗中的研究进展

诱骗寡聚脱氧核苷酸(decoy oligodeoxynucleotide,decoy ODN)通过与目的基因启动子上的转录因子结合位点(trascription factor binding site,TFBS)竞争性地结合转录因子,从而影响转录因子调控的靶基因表达。近年来,越来越多的研究者证实通过识别各种疾病相关的信号通路与转录因子,设计相应的decoy ODN,可以对一些疾病起到治疗效果,这其中包括运用STAT3 decoy ODN治疗恶性肿瘤和运用NF-κB decoy ODN治疗炎症纤维化疾病和心血管疾病。本文将总结分析近5年decoy ODN策略在疾病研究中的运用,为治疗肿瘤、纤维化、心脑血管病等疾病提供新的思路。

STAT3-Decoy ODN Inhibits Cytokine Autocrine of Murine Tumor Cells

Tumor cells usually secrete soluble factors to improve their proliferation via autocrine network or to escape from immune surveillance by inhibiting antitumor immunity, among these factors IL-10 and IL-6 play more important roles. Since both cytokines＇ signal transductions are mediated through the STAT3 pathway, STAT3 becomes an attractive target for tumor therapy. In present study, STAT3 of murine tumor cell lines B16 and MCA-38 was constitutively activated. After treatment with STAT3-decoy ODN, the proliferation of these tumor cells was inhibited and the transcription of IL-10 or IL-6 in tumor cells was down-regulated. These results suggested that STAT3 is a good target candidate, and STAT3-decoy ODN may possibly be used as a strategy for breaking both tumor autocrine network and tumor immunotolerance.

Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

Background：Decoy receptor 3 （DcR3） binds to Fas ligand （FasL） and inhibits FasL-induced apoptosis.The receptor is overexpressed in hepatocellular carcinoma （HCC）,and it is associated with the growth and metastatic spread of tumors.DcR3 holds promises as a new target for the treatment of HCC,but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3.The present work,therefore,examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2.Methods：HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3.After the knockdown of DcR3 was confirmed,cell proliferation,clone formation,ability of migrating across transwell membrane,and wound healing were assessed in vitro.Matrix metalloproteinase-9 （MMP 9） and vascular epithelial growth factor （VEGF）-C and D expressions of the DcR3 knockdown were also studied.Comparisons between multiple groups were done using one-way analysis of variance （ANOVA）,while pairwise comparisons were performed using Student＇s t test.P 〈 0.05 was regarded statistically significant.Results：DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2.Stable knockdown of DcR3 slowed down the growth of HepG2 （P 〈 0.05） and reduced the number of clones formed by 50% compared to those without DcR3 knockdown （P 〈 0.05）.The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control （P 〈 0.05） and suppressed the closure of scratch wound （P 〈 0.05）.In addition,the messenger RNA levels of MMP 9,VEGF-C,and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control （P 〈 0.05）.Conclusions：Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2.Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC.

Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor, fibroblast growth factor receptor-3, in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro. Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo. Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence. Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro. Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain, namely the substantia nigra, compact part, dorsal tier, substantia nigra and reticular part, but was not detected in the forebrain comprising the caudate putamen and striatum. Unusual results were obtained in retrosplenial locations of adult rat brain. We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses. We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8, a secretory factor. Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells. In contrast, addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons. Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1

Background： Decoy receptor 3 （DcR3） is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and tile metastasis of the tumor. This in vitro study aimed to investigate the effect ofdownregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. Methods： Three different cell lines were cultured： human cholangiocarcinoma TFK-I, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. Results： The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% （P 〈 0.01 ） and 48.03% （P 〈 0.05） of that of＇the control, respectively. It was found that the cell viability decreased to 61.87% of the control group （P 〈 0.01 ） after the downregulation of DcR3 in cholangiocarcinoma cell line TFK- 1 by transfection with DcR3-siRNA, while tile percentage ofapoptotic cells was 2.98 times as compared with the control group （P 〈 0.05）. Compared with the control group the ratio of G0/G1 increased, and the ratio of G2/M dccreased in the treatment group. However, the differences were not statistically significant. Conclusions： The effect of DcR3 on the growth and apoptosis ofcholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma.

Ischemic preconditioning protects against ischemic brain injury

In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.

Blood cellular mutant LXR-α protein stability governs initiation of coronary heart disease

AIM: To investigate the role of [breast and ovarian cancer susceptibility 1（BRCA1）-associated RING domain 1（BARD1）]/BRCA1 E3-ubiquitin ligase complex in governing the stability of mutant liver X receptor-（LXR-α） protein in coronary heart disease（CHD） subjects.METHODS: The expression analysis of various genes was carried out by quantitative real time polymerase chain reaction and western blotting within blood mononuclear cells of human CHD subjects at various stages of coronary occlusion and their corresponding normal healthy counterparts.Immunoprecipitation experiments were performed to establish protein interactions between LXR-αand BARD1.Peripheral blood mononuclear cells were cultured and exposed to Vitamin D3 and Cisplatin to validate the degradation of mutant LXR-αprotein in CHD subjects by BARD1/BRCA1 complex.RESULTS: The expression of mutant LXR-αprotein in CHD subjects was found to decrease gradually with the severity of coronary occlusion exhibiting a strong negative correlation,r =-0.975 at P 【 0.001.Further,the expression of BARD1 and BRCA1 also increased with the disease severity,r = 0.895 and 0.873 respectively（P 【 0.001）.Immunoprecipitation studies established that BARD1/BRCA1 complex degrades mutant LXR-αvia ubiquitination.The absence of functional LXR-αprotein resulted in increased expression of inflammatory cytokines such as interleukin（IL）-6,IL-8 and interferon-and decreased expression of ABCA1（ATP-binding cassette A1）（r = 0.932,0.949,0.918 and-0.902 with respect to Gensini score;P 【 0.001）.Additionally,cell culture experiments proved that Vitamin D3 could prevent the degradation of mutant LXR-αand restore its functional activity to some extent.CONCLUSION: Mutant LXR-αprotein in CHD subjects is degraded by BARD1/BRCA1 complex and Vitamin D3 can rescue and restore its function.

Response letter regarding the interpretation of gene expression data

This is a response letter to Verna E's comments regarding our previous manuscript published last year in the World Journal of Gastroenterology entitled "Relationship between LYVE-1,VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer",which evaluated the relationship between these expression levels and clinicopathological parameters(Ozmen F et al,World J Gastroenterology 2011;17:3220-3228).The mean values for lymphatic vessel endothelial hyaluronan receptor-1,CD44 and vascular endothelial growth factor receptor-3 expression(represented as 2-Ct) were 1.13,1.24 and 1.17,respectively,suggesting an increase in gene expression in tumor tissue compared to normal tissue.Despite the increase in gene expression in the cancer tissues(2-Ct > 1),only some of the results reached statistical significance,which was thoroughly discussed in our paper.In the present letter,we report that his comments are flawed and result in confusion.Therefore,we herein provide more explanation regarding gene expression in gastric cancer.We hope that this letter will address Verna E's misunderstandings.

Immunomagnetic Indirect Positive Sorting of Precartilaginous Stem Cells from Neonatal Rat

To investigate the technique of sorting high-purity precartilaginous stem cells from rat＇s perichondrium, neonatal rat＇s perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 （anti-FGFR-3）, and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody, Purity of the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 % -85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells from perichondrium cell suspensions.