AbstractThe overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3ʹ-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.

Polyphenol, one of the major components that exert the therapeutic effect of Chinese herbal medicine (CHM), comprises several categories, including flavonoids, phenolic acids, lignans and stilbenes, and has long been studied in oncology due to its significant efficacy against cancers in vitro and in vivo. Recent evidence has linked this antitumor activity to the role of polyphenols in the modulation of redox homeostasis (e.g., pro/antioxidative effect) in cancer cells. Dysregulation of redox homeostasis could lead to the overproduction of reactive oxygen species (ROS), resulting in oxidative stress, which is essential for many aspects of tumors, such as tumorigenesis, progression, and drug resistance. Thus, investigating the ROS-mediated anticancer properties of polyphenols is beneficial for the discovery and development of novel pharmacologic agents. In this review, we summarized these extensively studied polyphenols and discussed the regulatory mechanisms related to the modulation of redox homeostasis that are involved in their antitumor property. In addition, we discussed novel technologies and strategies that could promote the development of CHM-derived polyphenols to improve their versatile anticancer properties, including the development of novel delivery systems, chemical modification, and combination with other agents.

Methylmercury (MeHg) exposure during pregnancy can lead to adverse outcomes, including miscarriage and intrauterine growth retardation. In this study, MeHg cytotoxicity and its mechanisms in HTR-8/SVneo cells were investigated. MeHg inhibited HTR-8/SVneo cell viability and severely disrupted the cellular submicrostructure, showing a time-dose effect relationship. After MeHg treatment, the reactive oxygen species levels, malondialdehyde content, and superoxide dismutase (SOD) and catalase activities in the HTR-8/SVneo cells increased significantly with increased MeHg concentration (P<0.05). Similarly, MeHg also induced HTR-8/SVneo cell apoptosis in a dose-dependent manner. The proportion of cells in G1 phase decreased with increasing MeHg concentration, while that in the S and G2/M phases gradually increased. Moreover, cell migration and invasion capacities gradually decreased with increasing MeHg concentration, showing a significant difference between the MeHg-treated and control groups. Genes related to oxidative stress (HSPA6, HSPA1A, Nrf2, SOD1, HO-1, NQO1, OSGIN1, and gPX1), cell cycle (P21 and CDC25A), apoptosis (CYCS and AIFM2), and migration and invasion (CXCL8, CXCL3, CLU, IL24, COL3A1, MAPT, and ITGA7) were differentially expressed in the MeHg-treated group, indicating MeHg toxicity and mechanism of action. This study will provide insights into the prevention and treatment of pregnancy-related diseases caused by MeHg.

Background: Dry mouth sensation cannot be improved completely even though parotids are spared correctly. Our purpose is to develop a nomogram to predict the moderate-to-severe late radiation xerostomia for patients with locoregionally advanced nasopharyngeal carcinoma (LA-NPC) in intensity modulated radiation therapy (IMRT) / volumetric modulated arc radiotherapy (VMAT) era. Methods: A dataset of 311 patients was retrospectively collected between January 2010 and February 2013. The binary logistic regression was to estimate each factor's prognostic value for development of moderate-to severe patient-reported xerostomia at least 2 years (Xer2y) after completion of radiotherapy. Therefore, we can develop a nomogram according to binary logistic regression coefficients. This novel model was validated by bootstrapping analyses. Results: Contralateral Parotid mean dose (coMD<24.4Gy), VMAT (yes), and platinum-based concurrent chemoradiotherapy (no) were significantly related to patient-reported xerostomia at least 2 years (Xer2y) (all p < 0.001), and were included in the nomogram. Receiver operating characteristic (ROC) analysis revealed AUC (area under the ROC curve) with the value of 0.811 (0.710-0.912) of the nomogram, which was significantly higher than coMD 0.698 (0.560-0.840) from QUANTEC2010 (p<0.001). Calibration plots illustrated that the predicted Xer2y was close to the actual observation, and decision curve analyses (DCA) indicated valid positive net benefits. Conclusion: We developed a feasible nomogram to predict patient-rated Xer2y based on comprehensive individual data in patients with LA-NPC in the real world. The proposed model is able to facilitate the development of treatment plan and quality of life improvement.

Objective: To investigate the efficacy and safety of common clinical chemotherapy regimens in the treatment of elderly patients with AML, and to explore the effects of age stratification, gene mutation and leucocyte stratification on the survival of elderly patients with AML. Method: Part I: Collect all the literature published to January 2019 on the efficacy and adverse reactions of "3+7" regimen in the treatment of elderly AML patients compared with "3+7" regimen, or decitabine(DAC) combined with pre-excitation regimen. Evidence-based evidence is obtained by evaluating the efficacy and adverse reactions of "3+7" regimen compared with DAC regimen, pre-excitation regimen and DAC combined with pre-excitation regimen, respectively. The difference of efficacy and adverse reactions between the above schemes was confirmed. Part II: Retrospective analysis of clinical data of 90 elderly patients with AML (non-M3) hospitalized in the Department of Hematology, Sichuan Provincial People's Hospital from January 31, 2015 to January 31, 2019. These patients were divided into "3 + 7" group, the DAC group, and the DAC combined with the pre-excitation group according to the chemotherapy regimen. Complete response rate, partial response rate, and overall survival of each group were compared to evaluate the efficacy response. And explore the effects of genetic mutation, risk stratification, age stratification, ECOG score, white blood cell stratification on the survival of elderly AML. Results: Part I: A total of 12 articles were included, including 1715 elderly patients with AML. Through subgroup analysis, the results showed that the "3+7" regimen was superior to the DAC regimen in terms of CR; the infection of the "3+7" chemotherapy regimen was higher than that of the pre-excitation regimen. The mortality rate of "3+7" group was higher than that of pre-excitation group. Part II: The study included 90 patients, "3+7" group included 42 patients, DAC group included 12 patients, DAC combined pre-excitation group included 36 patients. In the three groups, the CR rate of the "3+7" chemotherapy group was 61.9%; the CR rate of the DAC group was 58.3%, while DAC combined with HAG or CAG treatment was 52.8%(P>0.05). In low risk group, the CR rate of the "3+7" chemotherapy regimen was 75%, the DAC group was 60%, while DAC+HAG/CAG was 75% (P=0.401). But the DAC+HAG/CAG group showed a survival advantage compared with the "3+7" group. There was no significant difference in survival between patients with FLT3-ITD mutations compared with those who without mutations. The OS of patients between 60 to 65 years were significantly better. The white blood cells were divided into <30×10^9/L group, 30-50×10^9/L group, 50-100×10^9/L group, and there was no difference in CR and OS in the group. About the time of neutropenia, the "3+7" chemotherapy regimen lasted for 15 (2-23) days, the DAC group was 11 (8-22) days, and the DAC+HAG/CAG was 16 (8-36) days, the difference between groups was between the DAC group and the DAC combined pre-excitation group (P=0.031). About platelet recovery time, "3+7" chemotherapy regimen lasted 12.5 (2-23) days, while DAC group lasted 11 (8-20) days and DAC+HAG/CAG group lasted 8 (5-16) days. Statistical differences could be observed in platelet recovery time between DAC group and DAC combined pre-excitation group (P=0.017). Lung infection rate was 83.3% in the "3+7" group, 77.8% in the DAC combined pre-excitation group, and the DAC group infection rate was 73.8%. 83.3%. Conclusion: Compared with other age groups, patients aged 60-65 have survival advantages. DAC-based drugs are more beneficial to patients than 3+7 chemotherapy. OS of patients without poor prognosis genome was significantly better than those with three or more poor prognosis genomes, while FLT3-ITD mutation had little effect on OS of elderly AML patients. The prognosis of patients with hyper-leucocyte is worse than that of patients without hyper-leucocyte, but WBC count has little effect on the long-term survival of patients with non- hyper-leucocyte. DAC group had obvious inhibitory effect on megakaryocytes. Disclosures Zheng: Pfizer: Research Funding.

Engineering malignant cells to express a heterologous α-gal antigen can induce heterograft hyperacute rejection, resulting in complement-dependent cytolysis (CDC) of tumor cells, which has been considered as a novel strategy for antitumor therapy. A549 cells engineered to express Galα1-3Galβ1-4GlcNAc-R (α-gal) epitope exhibited strong resistance to CDC treated by normal human serum (NHS) in a previous study. We hypothesized that the expression of membrane-bound complement regulatory proteins (mCRPs) decay accelerating factor (CD55) and protectin (CD59) influenced the efficacy of the α-gal/NHS-mediated antitumor effect to tumor cells in vitro. The present study confirmed that A549 cells expressed high levels of CD55 and CD59, whereas Lovo cells expressed relatively low levels of these proteins. A549 and Lovo cells transfected with plasmids containing or lacking the α-gal epitope were evaluated for their susceptibility to CDC by NHS and detected using a trypan blue exclusion assay. α-gal-expressing Lovo (Lovo-GT) cells were almost completely killed by α-gal-mediated CDC following incubation with 50% NHS, whereas no cytolysis was observed in α-gal expressing A549 (A549-GT) cells. Abrogating CD55 and CD59 function from A549-GT cells by various concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) or blocking antibodies increased the susceptibility of cells to CDC, and the survival rate decreased significantly comparing to the controls (P<0.05). The findings of the present study indicated that using the α-gal/NHS system to eliminate tumor cells via inducing the complement cascade reaction might represent a feasible approach for the treatment of cancer. However, high levels of mCRP expression may limit the efficacy of this approach. Therefore, an improved efficacy of cancer cell killing may be achieved by combining strategies of heterologous α-gal expression and mCRP downregulation.

Purpose: Cell cycle is mainly mediated by cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors. Cyclin E is the main regulator for transition from G1 to S phase, and is involved in cancer pathogenesis, progression and metastasis. Nevertheless, there is still a controversy of the prognostic value of cyclin E overexpression in ovarian cancer patients. This meta-analysis is the first study aimed at analyzing the effect of cyclin E overexpression on the prognosis of ovarian cancer.Methods: By systematically searching the PUBMED, EM BASE and MEDLINE databases for relevant articles with publication dates up to January 2016 and selection following inclusion and exclusion criteria, 8 studies with 1470 patients were enrolled in our meta-analysis. The overall survival (OS) of patients with cyclin E overexpression was calculated using hazard ratio (HR) with 95% confidence intervals (CIs). The studies were categorized according to the author and year, demographic data in each study, ovarian cancer related information, and cyclin E cut-off value.Results: Cyclin E overexpression in ovarian cancer was a poor prognostic factor with statistical significance for OS (HR=1.48, 95% CI: 1.12,1.85). Using confunnel, we found no publication bias in our analysis.Conclusion: Cyclin E might be considered as a prognostic factor for ovarian cancer, as supported by our meta-analysis. However, more high-quality studies should be conducted to find better clinical use of cyclin E in ovarian cancer.

Background and objectives: More observational studies from different parts of the world have been executed to demonstrate the molecular biological relationship between NGAL and colorectal cancer. Our previous study in 2014 has extracted data from 5 studies with literature screening until early 2013. This meta-analysis aimed to recruit more recent papers and explore the overall accuracy of NGAL detection on diagnosis. Materials and methods: We divided this meta-analysis into two layers: 1. distinguishing adenocarcinoma from other abnormalities and normal tissue; 2. distinguishing adenocarcinoma and abnormalities with high possibility of carcinogenesis from benign hyperplasia and normal tissue Results: For Layer 1, the pooled sensitivity and specificity of all studies were 0.81 (95% CI, 0.78-0.84) and 0.56 (95% CI, 0.52-0.60). The pooled positive likelihood ratio and negative likelihood ratio were 2.29 (95% CI, 1.40-3.74) and 0.34 (95% CI, 0.18-0.63). The pooled diagnostic odds ratios was 11.29 (95% CI, 3.36-38.01). The area under the summary receiver operating characteristic curve for the diagnosis of colorectal cancer was 0.8476. For Layer 2, the pooled sensitivity and specificity of all studies were 0.76 (95% CI, 0.73-0.79) and 0.63 (95% CI, 0.58-0.67). The pooled positive likelihood ratio and negative likelihood ratio were 7.11 (95% CI, 1.97-25.63) and 0.35 (95% CI, 0.23-0.54). The pooled diagnostic odds ratios was 23.10 (95% CI, 6.55-81.44). The area under the summary receiver operating characteristic curve for the diagnosis of colorectal cancer was 0.8900. Conclusion: NGAL is more suitable for screening adenocarcinoma and abnormalities with high possibility of carcinogenesis from benign hyperplasia and normal tissue.

BACKGROUND:High-quality cancer tissues are essential for future research, especially molecular research. For the sake of better quality of tissues, some storage methods are chosen according to lab conditions. But the impact of different storing conditions on the quality of RNA, DNA (especially the degree of DNA methylation), and protein of tissues that have undergone a thawing process, is not clear. METHODS:We analyzed the influence of different storage conditions including in RNALater solution, normal saline, Opti-mum Cutting Temperature compound (OCT), and snap frozen with no protective reagent (as control) in paired tissue samples on the quality of RNA (RNA Integrity Number value and mRNA expression), DNA quality (DNA amplification and DNA methylation degree of gene RASSF1a), and protein quality. Further, we analyzed the RNA quality of tissues that underwent three freeze-thaw cycles. RESULTS:The RNALater-treated group retained good RNA quality as expected on three repeated freeze-thaw cycles (RIN>8), but the snap-frozen tissues showed relatively poor results after one freeze-thaw cycle (RIN<7) and three times repeated freeze-thaw cycles (RIN<6). RNA from saline- and OCT-treated groups also yielded good results when we repeated freezing and thawing one time (RIN>7) and two times (RIN>6). The impact of different storing conditions on DNA amplification is small. However, DNA methylation and protein quality are different with different storing conditions. OCT seems to be more secure and stable compared with other two experimental groups, and show a similar trend with control group. CONCLUSIONS:In consideration of budget and efficiency, we suggest OCT as the best storing method that not only preserves RNA quality during the freezing-thawing process well, but also ensures more secure and stable DNA and protein.

Purpose LKB1 and FUS1 are two kinds of new tumor suppressor genes as well as early-stage genes in lung cancer. Recent studies showed that LKB1 and FUS1 play important roles in lung carcinogenesis process. We hypothesized that combined gene therapy with LKB1 and FUS1 could inhibit lung cancer growth and development synergistically. Methods In this study, two kinds of tumor suppressor genes, LKB1 and FUS1 , were constructed in an eukaryotic coexpression plasmid pVITRO 2 , and then, we evaluated the synergistic effects of the two genes on anticancer activity and explored the relevant molecular mechanisms. Results We defined coexpression of LKB1 and FUS1 could synergistically inhibited lung cancer cells growth, invasion and migration and induced the cell apoptosis and arrested cell cycle in vitro. Intratumoral administration of liposomes: pVITRO 2 – LKB1 – FUS1 complex (LPs–pVITRO 2 – LKB1–FUS1 ) into subcutaneous lung tumor xenograft resulted in more significant inhibition of tumor growth. Furthermore, intravenous injection of LPs–pVITRO 2 – LKB1–FUS1 into mice bearing experimental A549 lung metastasis demonstrated synergistic decrease in the number of metastatic tumor nodules. Finally, combined treatment with LKB1 and FUS1 prolonged overall survival in lung tumor-bearing mice. Further study showed that the synergistic anti-lung cancer effects of coexpression of LKB1 and FUS1 might be related to upregulation of p-p53, p-AMPK and downregulation of p-mTOR, p-FAK, MMPs, NEDD9, VEGF/R and PDGF/R. Conclusions Our results suggest that combined therapy with eukaryotic coexpression plasmid carrying LKB1 and FUS1 genes may be a novel and efficient treatment strategy for human lung cancer.

Engineering cancer cells to express heterologous antigen α-gal and induce the destruction of tumor cells depending on the complement cascade may be a promising strategy of tumor therapy. However, the feasibility and effect of using α-gal to induce colorectal adenocarcinoma cell line cytolysis is not yet known. In this study, we evaluated α-gal expression's ability to sensitize human colorectal adenocarcinoma cell lines to complement attack in cell lines LoVo, SW620, and Ls-174T. Nearly all α-gal-expressing LoVo and SW620 cells were killed by normal human serum (NHS), but α-gal-expressing Ls-174T cells showed no significant lysis. We analyzed the expression levels of membrane-bound complement regulatory proteins (mCRPs) on the three cell lines, and their protective role in α-gal-mediated activation of the complement. LoVo showed no expression of any of the three proteins. CD59 was strongly expressed by SW620 and Ls-174T. CD46 and CD55 varied between the two cell lines. CD46 on SW620 was only half the intensity of CD46 on Ls-174T. Ls-174T showed a notable expression of CD55, while expression of CD55 on SW620 was not detected. The sensitivity of Ls-174T expressing α-gal to NHS greatly increased following the downregulation of CD46 and CD55 with short hairpin RNA (shRNA). However, there is no increase in cell killing when CD59 expression was diminished. Our findings suggest that the use of α-gal as antigen to induce tumor cell killing may be a potential therapeutic strategy in colon cancer and that CD55 plays a primary role in conferring resistance to lysis.

BACKGROUND AND OBJECTIVE:The iNOS gene is associated with NO-mediated antitumor effects. The aims of this study are to construct a eukaryotic expression plasmid that carries the iNOS gene and to detect the expression levels and antitumor effects of the iNOS gene on A549 lung cancer cells.METHODS:A DNA fragment of the human iNOS coding sequence was amplified using reverse transcription polymerase chain reaction (RT-PCR). The DNA fragment was subsequently cloned into the multiple cloning sites of the eukaryotic expression vector pVAX. The recombinant plasmid was confirmed using restriction enzyme treatment, PCR, and sequencing and was then transfected into A549 lung cancer cells. The expression of the iNOS gene in the A549 lung cancer cells after transfection was verified by RT-PCR and Western blot analysis. The effects of iNOS on cell apoptosis, proliferation, and migration were identified by staining with Hoechst 3235, an MTT assay, and a scratch assay, respectively.RESULTS:The results of the restriction enzyme digestion, PCR, and sequencing verified the successful construction of the eukaryotic expression plasmid pVAX-iNOS. The iNOS gene expression level was increased in the transfected A549 cells. Further experiments also showed increased cell apoptosis among the A549 lung cancer cells transfected with pVAX-iNOS. Meanwhile, the proliferation and migration of A549 cells were significantly inhibited by the enhanced iNOS gene expression.CONCLUSION:The recombinant eukaryotic expression vector pVAX-iNOS was successfully constructed and transfected into A549 cells. The enhanced iNOS gene expression significantly promoted cell apoptosis, whereas the proliferation and migration of A549 cells were inhibited. These findings contribute to the development of novel and effective gene therapies for lung cancer.

This experimental study was aimed to construct the recombinant bisbicistronic eukaryotic expression vector containing endocrine and exocrine protein (EECP) gene associated with breast cancer and enhanced green fluorescent protein (EGFP) gene. And then we transfected it into breast cancer cells MCF-7 to detect the expression of EECP protein and study preliminary biological function of EECP gene. The EECP sequence was cloned to pBluescript II SK (+) plasmid. After restriction endonuclease reaction of pBluescript II SK(+) plasmid, the EECP fragment was cloned to pIRES2-EGFP vector forming a recombinant eukaryotic expression vector named pEECP-IRES2-EGFP. The potential vector was identified by restriction endonuclease digestion and sequencing. Correct plasmid was extracted and transfected into breast cancer cells MCF-7. The expression of EECP protein was detected by western blot analysis. Its biological function was studied by MTT and Flow-cytometry. It turns out that the recombinant eukaryotic expression vector containing EECP gene and EGFP gene was constructed successfully, and it could transfect MCF-7 cells efficiently. It can get higher expression of EECP protein and higher cell proliferation, thus providing an important and convenient tool for studying the function of EECP gene in vitro and in vivo.

Cisplatin is one of the most effective antitumor drugs for non-small cell lung carcinoma (NSCLC) patients. However, its efficacy has encountered a plateau due to its side effects and drug resistance. Inducible nitric oxide (NO) synthase (iNOS) gene therapy has been reported to have antitumor effects in several types of cancers and enhances sensitivity to cisplatin, but the effects of iNOS gene therapy alone or its combination with cisplatin in lung cancer remain unclear. In the current study, we evaluated the effects of cationic liposome (LP)-mediated iNOS gene transfection on enhancing low-dose cisplatin-mediated antitumor effects in the A549 human lung adenocarcinoma cell line in vitro. Furthermore, we examined whether iNOS gene therapy enhances the antitumor effects of low-dose cisplatin in two A549 human lung cancer cell xenograft mouse models. The results revealed that iNOS gene therapy may significantly enhance low-dose cisplatin-mediated inhibition of cell proliferation, invasion, migration and promotion of cell apoptosis in A549 cells. Intratumoral administration of the LP-pVAX-iNOS complex significantly enhanced low-dose cisplatin-mediated suppression of subcutaneous tumor growth. Moreover, intravenous injection of the LP-pVAX-iNOS complex greatly enhanced low-dose cisplatin-mediated inhibition of experimental lung metastasis and prolonged the life span of mice without significant organ-related toxicity in a nude mouse model of lung metastasis compared to the cisplatin alone-treated group. Furthermore, iNOS gene-mediated enhancement of cisplatin-mediated antitumor effects in lung cancer may be related to the attenuation of p-mTOR, MMP2 and the activation of p-p53. Thus, the combination treatment with iNOS gene therapy and cisplatin may be a novel and effective therapeutic strategy for lung cancer.

OBJECTIVE:To investigate the clinical significance of tumor microvascular density (MVD) detected by anti-CD105 and anti-CD34 as a predictor of recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT). METHODS:One hundred and twelve (100 with adjacent nontumorous area) patients with HCC who underwent LT between Jan. 2001 and Dec. 2006 were included in this retrospective study. Paraffin blocks of tumor tissue and adjacent nontumorous tissue were used for immunohistochemical study. MVD of HCC was evaluated by anti-CD105 monoclonal antibody and anti-CD34 monoclonal antibody. RESULTS:The immunohistochemich staining showed CD34 strongly positive expressed in the tumor area with tumor mature vasculature, and CD105 strongly positive expressed in adjacent nontumorous area with tumor newly formed vessels. Univariate analysis using chi-square test showed portal vein tumor thrombus (PVTT), pTNM stage, MVD-CD105 expression in adjacent nontumorous area, tumor size and serum AFP level were significantly associated with HCC recurrence after LT (P < 0.05). Multivariate analysis by Cox's regression model showed MVD-CD105 expression in adjacent nontumorous area and PVTT still remained significant correlation with recurrence of HCC after LT (P < 0.05), and MVD-CD105 in adjacent nontumorous area was significantly correlated with PVTT (r(s) = 0.257, P = 0.01), pTNM stage (r(s) = 0.350, P = 0.000), as well as serum AFP level (r(s) = 0.208, P = 0.038). CONCLUSION:The anti-CD105 mAb is an ideal tool to quantify new microvessels in HCC. MVD-CD105 expression in adjacent nontumorous area may be used as an additional factor for the identification of patients at risk for post-transplant recurrence.

Aim: To investigate the anti-inflammatory effect of Z-ligustilide (LIG) on lipopolysaccharide (LPS)-activated primary rat microglia. Methods: Microglia were pretreated with LIG 1 h prior to stimulation with LPS (1 μg/mL). After 24 h, cell viability was tested with MTT, nitric oxide (NO) production was assayed with Griess reagent, and the content of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein (MCP-1) was measured with ELISA. Protein expression of the nuclear factor-κB (NF-κB) p65 subunit, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was detected with immunocytochemistry 1 h or 24 h after LPS treatment. Results: LIG showed a concentration-dependent anti-inflammatory effect in LPS-activated microglia, without causing cytotoxicity. Pretreatment with LIG at 2.5, 5, 10, and 20 μmol/L decreased LPS-induced NO production to 75.9%, 54.4%, 43.1%, and 47.6% ( P <0.05 or P < 0.01), TNF-α content to 86.2%, 68.3%, 40.1%, and 39.9% ( P <0.01, with the exception of 86.2% for 2.5 μmol/L LIG), IL-1β content to 31.5%, 27.7%, 0.6%, and 0% ( P <0.01), and MCP-1 content to 84.4%, 50.3%, 45.1%, and 42.2% ( P <0.05 or P <0.01), respectively, compared with LPS treatment alone. LIG (10 μmol/L) significantly inhibited LPS-stimulated immunoreactivity of activated NF-κB, COX-2, and iNOS ( P <0.01 vs LPS group). Conclusion: LIG exerted a potent anti-inflammatory effect on microglia through inhibition of NF-κB pathway. The data provide direct evidence of the neuroprotective effects of LIG and the potential application of LIG for the treatment of the neuroinflammatory diseases characterized by excessive microglial activation.

OBJECTIVES To establish a human lung adenocarcinoma cell subline A549 that can stably express the Chinese Banna minipig inbred-line (BMI) alpha1 ,3-galactosyltransferase (alpha1 ,3GT) gene and alpha-galactosyl (Gala1-3Galb1-4GlcNAc-R, alpha-gal) epitopic, providing a cell model which expressed xenotransplantation antigens for the further research on the effect of complement dependent cytotoxic lysis of the tumor cells triggered by human natural serum. METHODS The pEGFP-CMV-GT plasmid containing Banna minipig alpha1 ,3-GT gene was ransfected into A549 cells with lipofectin in vitro. After screened with G418,the single clones were got out and then amplified, the stable transfected cells was named A549-GT. The transcription of alpha1, 3-GT gene in A549-GT cells was detected by RT-PCR. Direct immunofluonrescence methods and flow cytometer were performed to observe the expression of alpha-gal and the binding conditions of IgM and complement C3 in human serum on A549-GT cells. The biological characters of A549-GT cells including morphology, proliferation, and tumorigenesis in nude mice were also examined. RESULTS After G418 screening, A549-GT that stablely transfected with alpha1, 3-GT gene was obtained and has been passaged for 2 years. The expression of alpha1,3-GT mRNA and alpha-gal was detected continuously and stably in A549-GT. The expression rate of alpha-gal positive cells reached 80.1% +/- 3.2%. The binding of human serum IgM and C3 in human serum on A549-GT cells were founded. Compared with parental A549 cells, its biological characteristics did not change. CONCLUSION A549-GT cell line stably and continuously expressing alpha1, 3-GT and alpha-gal was established successfully. It provided a useful cell model for the further study of pig alpha1,3-GT gene in tumor immunotherapy.

Objective:To construct an xenoantigen synthetase α-1,3 galactosyltransferase(α-1,3GT) eukaryotic expression vector regulated by human telomerase catalytic subunit(hTERT) promoter,and to investigate its targeting expression of α-1,3GT in lung cancer cell lines.Methods:Previously prepared and confirmed pig α-1,3GT gene was inserted into pEGFP-hTERTp plasmid to construct eukaryotic expression vector pEGFP-hTERTp-GT.pEGFP-hTERTp-GT and pEGFP-N1-GT(α-1,3GT eukaryotic expression vector under the control of CMV promoter) were transfected into telomerase-positive human lung adenocarcinoma A549 cells and telomerase-negative human embryonic lung fibroblast MRC-5 cells.1,3-GT mRNA expression in the transfected cells was detected by RT-PCR.Expression of α-gal antigen in transfected cells was examined by immunofluorescence and flow cytometry.Results:pEGFP-hTERTp-GT eukaryotic expression vector was successfully constructed.Both A549 and MRC-5 cells transfected with pEGFP-N1-GT showed expression of α-1,3GT mRNA;A549 cells but not telomerase-negative MRC-5 cells expressed α-1,3GT mRNA after transfection with pEGFP-hTERTp-GT.Furthermore,both A549 and MRC-5 cells transfected with pEGFP-N1-GT showed expression of xenoantigen α-gal;A549 but not MRC-5 cells expressed xenoantigen α-gal after transfection with pEGFP-hTERTp-GT(P0.01).Conclusion:α-1,3GT gene under the regulation of hTERT promoter can be specifically expressed in telomerase-positive lung cancer cell lines,which can induce production of xenoantigen α-gal.

OBJECTIVE:To study the expression rule of proliferating cell nuclear antigen (PCNA) in thyroid when the hyperplasia of male rat thyroid is induced by propylthiouracil (PTU).METHODS:PTU was administered to rats by gavage at a dose of 5.0 mg/kg B. W for 0,3,6,9 and 12 days respectively. All animals were sacrificed after the last dosage, and the expression of PCNA in thyroid was detected by immunohistochemistry and RT-PCR.RESULTS:Compared with the control group, the PCNA level in the thyroid of rats given PTU for 3 days significantly increased (P < 0.05),and reached the highest expression when it was 6 days, but then when it was 9 days and 12 days, the PCNA expression showed a descending tendency.CONCLUSION:PCNA has a expression rule in hyperplastic thyroid: expression increase first but then decrease. This rule confirms the histological change of thyroid in earlier stage. The experimental time of assessment test to thyroid hormone disruptors will be shortened to six days.