Interleukin-12 inhibits the hepatocellular carcinoma growth by inducing macrophage polarization to the M1-like phenotype through downregulation of Stat-3.

Hepatocellular carcinoma is the third most common cause of cancer death worldwide. Novel early detection biomarkers and efficacious therapy strategies are needed. Macrophages recruited from circulation monocytes are the major component of solid cancer and play an important role in the carcinogenesis. Whether overexpression of L-12 in monocytes could induce the phenotype directional differentiation into tumoricidal M1 macrophages and inhibit HCC growth in tumor microenvironment was investigated in this study. For the establishment of the monocyte/IL-12 and polarization of M1-like macrophage, the IL-12 overexpressing recombinant monocyte/IL-12 cells were established by infecting with pAd5F35-CMV/IL-12 adenovirus and co-cultured with HCC SMMC-7721 and Hep3B cells. It was found that the phenotype of monocyte/IL-12 polarized to M1-like macrophages with CD197high IL-12high CD206low IL-10low, and decreased expression of TGF-ß, VEGF-A, and MMP-9. In order to explore the mechanism underlying the macrophages polarization, we detected the Stat-3 pathway and its downstream transcription factor c-myc, and found that the p-Stat-3 and c-myc were down-regulated. To evaluate the effects of monocyte/IL-12 on inhibiting HCC growth, various assays including CCK8, flow cytometry, colony-forming and Transwell assays in vitro, and xenograft mouse models and immunohistochemical analyses in vivo were used to detect the HCC growth and relative markers. Treated with IL-12 overexpressing monocytes, the xenograft tumor growth was significantly inhibited in vivo. These results have proven that IL-12-overexpressed monocytes could directionally differentiate to M1-like macrophages through downregulation of Stat-3 and result in the inhibition of HCC growth.

IL-12 could induce monocytic tumor cells directional differentiation.

Interleukin-12 (IL-12), a member of interleukin family, plays a critical role in immune responses and anti-tumor activity. In this study, the effects of IL-12 on monocytic tumor cell lines differentiation to macrophagocyte and its likely mechanism was investigated. We examined the differentiation markers, morphological and functional changes, and possible mechanism in IL-12-treated THP-1 and U937 cells. It was found that IL-12 could up-regulated macrophage surface marker CD68 and CD11b expression in a time-dependent manner. Morphologically, after IL-12 treatment, THP-1 and U937 cells became round or irregular shape, even stretched many cell membrane protuberances; some cell nuclei became fuzzy or completely disappeared, and the chromatin appeared dense and cordlike. Furthermore, IL-12-induced monocytic tumor cell differentiation was accompanied by the growth arrest with G1-phase accumulation and S-phase reduction; apoptosis increased with anti-apoptosis protein Bcl-2 down-expression and pro-apoptosis protein Fas up-regulation, and enhanced phagocytosis function. The IL-12-induced macrophage differentiation of THP-1 and U937 cells was associated with the up-regulation of c-fms expression and the CSF-1R Tyr 809 site phosphorylation. These findings have revealed that IL-12 could induce monocytic tumor cells directional differentiation into macrophage-like cells, and its mechanism is possible connected with the up-regulation of c-fms expression and the phosphorylation of CSF-1R Tyr-809 site.

IL-12 regulates B7-H1 expression in ovarian cancer-associated macrophages by effects on NF-κB signalling.

BACKGROUND AND AIM: B7-H1, a co-inhibitory molecule of the B7 family, is found aberrantly expressed in ovarian cancer cells and infiltrating macrophage/dendritic-like cells, and plays a critical role in immune evasion by ovarian cancer. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects on ovarian cancer. However, whether IL-12 regulates B7-H1 expression in human ovarian cancer associated-macrophages has not been clarified. Therefore, we investigated the effects of IL-12 on the expression of B7-H1 in ovarian cancer-associated macrophages and possible mechanisms. METHODS: PMA induced THP-1-derived macrophages or human monocyte-derived macrophages were treated with recombinant IL-12 (rIL-12) or infected with adenovirus carrying human IL-12 gene (Ad-IL-12-GFP) for 24 h, then cocultured with the SKOV3 ovarian cancer cell line for another 24 h. Macrophages were collected for real-time PCR and Western blot to detect the expression of B7-H1, and activation of the NF-κB signaling pathway. Moreover, supernatants were collected to assay for IL-12, IFN-γ and IL-10 by ELISA. In addition, monocyte-derived macrophages treated with IFN-γ were cocultured with SKOV3 and determined for the expression of B7-H1. Furthermore, the expression of B7-H1 in monocyte-derived macrophages was also evaluated after blocking NF-κB signaling. RESULTS: The expression of B7-H1 was significantly upregulated in monocyte-derived macrophages treated with rIL-12 or Ad-IL-12-GFP compared with the control groups (p<0.05), accompanied by a remarkable upregulation of IFN-γ (p<0.05), a marked downregulation of IL-10 (p<0.05) and activation of NF-κB signaling. However, the upregulation of B7- H1 was inhibited by blocking the NF-κB signaling pathway (p<0.05). Expression of B7-H1 was also increased (p<0.05) in monocyte-derived macrophages treated with IFN-γ and cocultured with SKOV3. By contrast, the expression of B7-H1 in THP-1-derived macrophages was significantly decreased when treated in the same way as monocyte-derived macrophages (p<0.05), and IL-10 was also significantly decreased but IFN-γ was almost absent. CONCLUSIONS: IL-12 upregulates the expression of B7-H1 in monocyte-derived macrophages, which is possible though inducing the secretion of IFN-γ and further activating the NF-κB signal pathway. However, IL-12 downregulates the expression of B7-H1 in THP-1-derived macrophages, associated with a lack of IFN-γ and inhibition of expression of IL-10.

Integrated analysis of long non-coding RNAs and mRNA expression profiles reveals the potential role of lncRNAs in gastric cancer pathogenesis.

Long non-coding RNAs (lncRNAs) have been shown to play a critical role in cancer biology and are frequently aberrantly expressed. Despite their important role in pathology, little is known mechanistically regarding their role in gastric cancer (GC) pathogenesis. To characterize the role of lncRNAs in GC pathogenesis, 8 paired human GC tissue samples and matched adjacent normal tissue were examined. Large scale expression profiling of lncRNA and mRNA was performed utilizing microarray technology and validated by qPCR. Differentially expressed lncRNAs were subjected to bioinformatic analysis to predict target genes, followed by the integration of differentially expressed mRNA data and GO and network analysis to further characterize potential interactions. In our study, 2,621 lncRNAs and 3,121 mRNAs were identified to be differentially expressed (≥2.0-fold change) in GC samples relative to their matched counterparts. lncRNA target prediction revealed the presence of 221 potential lncRNA-mRNA target pairs for the 75 differentially expressed lncRNAs and 60 differentially expressed genes. KEGG pathway analysis showed that these target genes were significantly enriched in 7 different pathways, of which the p53 signaling pathway was the most significant and has been previously implicated in GC pathogenesis. Construction of a lncRNA-mRNA correlation network revealed 10 differentially expressed lncRNAs potentially regulating the p53 signaling pathway. Overall, this is the first study perform global expression profiling of lncRNAs and mRNAs relating to GC. These results may provide important information for further insights into the pathogenesis of GC and provide potential targets for future therapeutics.

Fused polypeptide with DEF induces apoptosis of lung adenocarcinoma cells.

To analyze the effects of a new unknown peptide DEF on the growth of tumor cells, a fused polypeptide TAT-DV1-DEF was designed and synthesized. The lung adenocarcinoma cell line GLC-82 treated with TAT- DV1-DEF was analyzed with a cell counting kit 8, and the location of polypeptides in cells was observed under laser confocal microscopy. The efficiency of polypeptide transfection and changes in nuclear morphology were analyzed by flow cytometry and fluorescence microscopy, respectively. Finally, the mechanism of tumor cell growth inhibition was evaluated by Western blotting. We found that TAT-DV1-DEF could significantly inhibit the growth of the lung adenocarcinoma cell line GLC-82, but not the normal human embryonic kidney cell line HEK-293. Polypeptides were found to be mostly localized in the cytoplasm and some mitochondria. The efficiency of polypeptide transfection in the two cell types was approximately 99%. Apoptotic nuclei were observed under fluorescence microscopy upon treatment with polypeptides and DAPI staining. Western blot analyses indicated that the polypeptide inhibition of tumor cell growth was apoptosis dependent. In the present study, we demonstrated that fused polypeptides could induce apoptosis of the lung adenocarcinoma cell line GLC-82, indicating that the new unknown peptide DEF has antitumor effects.

[Prokaryotic expression and purification of a tandem repeat of ovarian cancer antigen CA125 and preparation of its antiserum].

AIM: To establish a prokaryotic expression system of the tandem repeat of CA125 (CA125R), express and purify the recombinant CA125R protein, prepare its antiserum. METHODS: The full gene sequence of one tandem repeat of CA125 was synthesized and cloned into pET-32a(+) to construct a prokaryotic expression vector of the CA125R protein (pET-CA125R). The pET-CA125R was transformed into E.coli BL21 (DE3) and the soluble expression conditions were optimized; the pure recombinant CA125R protein was prepared by affinity Ni-NTA chromatography and identified by Western blotting. A rabbit was immunized with the pure recombinant CA125R protein to prepare its antiserum. RESULTS: The prokaryotic expression vector of CA125R was successfully constructed. The optimal soluble induction expression conditions were 0.5 mmol/L isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 15DegreesCelsius for 6 h. Western blotting confirmed the pure CA125R recombinant protein of high purity. The prepared antiserum specifically recognized recombinant CA125R protein and natural CA125 glycoprotein. CONCLUSION: We successfully established the efficient prokaryotic expression system of the CA125R, and prepared the recombinant CA125R protein of high purity and its antiserum.

¹H NMR-based serum metabolic profiling in compensated and decompensated cirrhosis.

AIM: To study the metabolic profiling of serum samples from compensated and decompensated cirrhosis patients. METHODS: A pilot metabolic profiling study was conducted using three groups: compensated cirrhosis patients (n = 30), decompensated cirrhosis patients (n = 30) and healthy controls (n = 30). A ¹H nuclear magnetic resonance (NMR)-based metabonomics approach was used to obtain the serum metabolic profiles of the samples. The acquired data were processed by multivariate principal component analysis and orthogonal partial least-squares discriminant analysis (OPLS-DA). RESULTS: The OPLS-DA model was capable of distinguishing between decompensated and compensated cirrhosis patients, with an R²Y of 0.784 and a Q²Y of 0.598. Twelve metabolites, such as pyruvate, phenylalanine and succinate, were identified as the most influential factors for the difference between the two groups. The validation of the diagnosis prediction showed that the accuracy of the OPLS-DA model was 85% (17/20). CONCLUSION: ¹H NMR spectra combined with pattern recognition analysis techniques offer a new way to diagnose compensated and decompensated cirrhosis in the future.

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species.

We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.

Use of duplex mutation primers for real-time PCR quantification of hepatitis C virus RNA in serum.

BACKGROUND: The duplex mutation primers offer many advantages over other multi-labeled probes for real-time detection of amplification products. OBJECTIVES: To develop and validate a novel real-time PCR for quantification of HCV RNA based on the duplex mutation primers technology. MATERIALS AND METHODS: The duplex mutation primers were selected in the highly conservative 5' non-coding region (5'NCR) of the HCV RNA. The assay was validated with the Viral Quality Control panel, which also includes Chinese HCV RNA standards. RESULTS: The detection limit was 57 IU/mL, and a good linear correlation in the range of 102-108 IU/mL was revealed (r(2) = 0.999) with the novel method. This assay has a dynamic range of at least 8 log10 without the need for specimen dilution, good clinical intra- and inter-run precision, and excellent correlation with a commercially available assay(r(2) = 0.95). CONCLUSIONS: The high sensitivity, wide linear range, and good reproducibility, combined with low cost, make this novel quantitative HCV real-time PCR assay particularly well suited for application to clinical and epidemiological studies.

[CpG array analysis of histone H3 lysine 4 trimethylation in patients with IgA nephropathy].

OBJECTIVE: To investigate the aberrance of histone H3 lysine 4 trimethylation (H3K4me3) in patients with IgA nephropathy (IgAN). METHODS: In 15 patients with IgAN and 15 healthy volunteers, H3K4me3 variations in peripheral blood mononuclear cells (PBMCs) were analyzed using chromatin immunoprecipitation and microarray analysis (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Quantitative real-time PCR (qRT-PCR) was carried out to examine the correlations between the mRNA expression profiles and H3K4me3 levels. RESULTS: We identified 83 genes that displayed significant H3K4me3 differences in IgAN patients compared with healthy subjects. Among them, 39 genes showed increased H3K4me3 and 44 genes had decreased H3K4me3 levels. The results of ChIP real-time PCR were well consistent with the microarray data. Quantitative RT-PCR revealed the correlations between the mRNA expressions and the methylation levels of H3K4me3. CONCLUSION: IgAN patients have significant alterations in H3K4me3, and the genes with aberrant H3K4me3 may provide insights into the pathogenesis of IgAN.

Elevated ß-arrestin1 expression correlated with risk stratification in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the main subtype of childhood leukemia. Risk stratification is pivotal for ALL prognosis and individualized therapy. The current factors for risk stratification include clinical and laboratory features, cytogenetic characteristics of the blast, early response to chemotherapy, and genetic factors. Analyses of gene expression are becoming increasingly important in ALL risk stratification. ß-Arrestin1, a multifunctional scaffold protein mediating many intracellular signaling networks, has been shown to be involved in many tumors. However, little is known of ß-arrestin1 in leukemia. In this study, we found that ß-arrestin1 was significantly elevated in 155 newly diagnosed ALL patients, compared with 51 controls. Further analysis showed that ß-arrestin1 expression was positively related with risk classification and white blood cell count in ALL. Moreover, expression of Notch1, an essential gene for developing hematological cells and T-ALL, was found to be negatively correlated with ß-arrestin1 in ALL. In conclusion, ß-arrestin1 may be a useful predictor of risk stratification and prognosis of ALL, and thus of potential use in the design of individualized therapy strategies.

[The study of the differential gene expression profiles related to toxic effects in rats exposed to silica].

OBJECTIVE: To study the differential gene expression profiles related to toxic effects in rats exposed to silica. METHODS: Wistar rats exposed to SiO2 (50 mg/ml) and 1 ml normal saline by intratracheal injection served as the exposure and control groups, on the 14th day after exposure all rats were executed and the rat lung tissues were obtained. The differential gene expression profiles in the lung tissues of rats exposed to silica were detected using confocal fiber beads gene chip technique, and the differential expression profiling data were analyzed using the database for annotation, visualization and integrated discovery (DAVID) bioinformation analysis tool. RESULTS: The results of present study indicated that 1567 genes with differential expression were identified in 22107 genes of rat lung tissues in exposure group, including 765 up-regulated genes and 802 down-regulated genes as compared to control group. In the 461 genes related to toxic effects, 285 genes were up-regulated and 176 genes were down-regulated in exposure group. The trends of up-regulation of HMOX1 and SOD2 genes in RT-PCR assay were similar to those in gene chip technique. CONCLUSION: A large number of genes related to toxic effects in the rats with silica-induced pulmonary fibrosis appeared up-regulation or down-regulation. There may be a complex gene regulation network in the pulmonary fibrosis induced by SiO2, and the toxicological mechanism is an important part in the development of pulmonary fibrosis.

[Predictive value of human fatty acid binding protein for myocardial ischemia and injury in perioperative period of cardiac surgery].

OBJECTIVE: To evaluate the value of human fatty acid binding protein (h-FABP) in predicting myocardial ischemia and injury in the perioperative period of cardiac surgery, we observed the dynamic changes of h-FABP in perioperative period of patients underwent coronary artery bypass grafting and ventricular septal defects repairing surgery, and evaluated the relationship of h-FABP and ischemia modified albumin (IMA), CK-MB, cTnI. METHODS: Patients underwent coronary artery bypass grafting (n=30) and ventricular septal defect repairing (n=30) surgery between February 2008 and December 2008 were included in this study. Venous blood sample was obtained at preoperative, aortic clamping, aortic unclamping of 10 min, 2 h, 6 h, 12 h, 24 h for the measurements of h-FABP, IMA, cTnI and CK-MB. RESULTS: h-FABP and IMA changed in the same way at various examined time points, h-FABP changes also paralleled cTnI and CK-MB changes, h-FABP peaked early during myocardial ischemia and injury and returned to baseline level at 2 h post myocardial ischemia and injury. Linear correlation analysis showed that the peak value of h-FABP was positively correlated with IMA, CK-MB and cTnI in both CABG group (r = 0.948, 0.964 and 0.961, P < 0.05) and in the VSD group (r = 0.986, 0.978 and 0.957). CONCLUSIONS: h-FABP is an early diagnostic parameter reflecting perioperative myocardial ischemia and injury in cardiac surgery. Quantitative h-FABP monitoring could predict the severity of myocardial ischemia and injury early during cardiac surgery.

[Effect of hepatocyte growth factor gene transfection on biological features of lymphoma cells].

OBJECTIVE: To investigate the biological effect of hepatocyte growth factor (HGF) on HGF gene-transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue, HGF gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF. The recombinant vector was transfected to Raji cells, and the stably transfected cells were selected by homomycin B in serial passages, and confirmed by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry. The biological features of transfected Raji cells were evaluated by semisolid culture. RESULTS: RT-PCR results showed that Raji cells were transfected successfully with recombinant eukaryotic expression vector pVITRO2-mcs-HGF. HGF mRNA and protein were expressed successfully in Raji cells. Expression of HGF gene enhanced proliferation, metastasis and invasion of Raji cells. CONCLUSION: HGF gene has been cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF successfully. Transfected HGF may change the biological features of Raji cells.

[Cytogenetic and molecular characterization of partial trisomy 4q and partial monosomy 10q in a patient].

OBJECTIVE: To ascertain the karyotype of a girl with moderate mental retardation and growth retardation, perform correlation analysis between chromosomal variation and phenotype, and investigate the application and superiority of array-based comparative genomic hybridization (array-CGH) in clinical cytogenetic diagnosis. METHODS: G-banded chromosome analysis, array-CGH, fluorescence in situ hybridization (FISH) and real-time quantitative PCR (RQ-PCR) were used to ascertain the karyotype of the patient and her relatives. RESULTS: G-banding analysis of the patient showed a derivative chromosome 10 with an extra fragment on its long arm terminal, both her father and grandmother had an apparently balanced translocation t(4;10)(q25;q26). Array-CGH revealed that the breakpoint on chromosome 4 was located at 4q26. In addition, a microdeletion of about 0.54 Mb del(10)(q26.3) was identified from the patient. FISH and RQ-PCR confirmed that the del(10)(q26.3) was also present in both her father and grandmother. CONCLUSION: No recognizable phenotype was associated with del(10)(q26.3). The abnormal phenotypes presented in the patient may be ascribed to the 4q26-q35.2 triplication. Further more, compared with conventional cytogenetic analysis, array-CGH is of high resolution and high accuracy.

Macrophage-specific overexpression of antimicrobial peptide PR-39 inhibits intracellular growth of Salmonella enterica serovar Typhimurium in macrophage cells.

Although purified and synthesised PR-39 shows potent antibacterial effects in vitro, its ability to kill intracellular bacteria in macrophages, which are a major cause of refractory intracellular infection, has not yet been demonstrated. Both to enhance its antimicrobial potential and to reduce systemic side effects, it would be desirable to deliver PR-39 into macrophage cells and to limit its activation to the site of infection. To address this issue, PR-39 DNA was inserted into the eukaryotic expression plasmid pIRES2-EGFP and the adenoviral vector Ad-MSP, from which PR-39 can be specifically expressed in macrophage cells from the macrophage-specific promoter. pIRES2-EGFP/PR39 and Ad-MSP/PR-39 were either transduced or infected into macrophage RAW264.7 cells for stable or transient expression of PR-39. PR-39 expression in macrophage cells was subsequently confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. Furthermore, its antimicrobial activity in macrophage cells was evaluated by colony enumeration assay. Results showed that the macrophage-specific promoter could initiate targeted expression of PR-39 only in macrophage RAW264.7 cells. Moreover, either stable or transient expression of PR-39 in macrophage cells conferred enhanced antimicrobial activity against Salmonella enterica serovar Typhimurium. Our results have demonstrated that macrophage-specific expression of antimicrobial peptide PR-39 in macrophages could inhibit the growth of intracellular S. Typhimurium and indicated it to be a novel and promising approach for the control of refractory intracellular infection.

[The study on differential gene expression profiling in pulmonary tissue of rats exposed to silica early].

OBJECTIVE: To study the differential gene expression profiling of rats exposed to silica using the normal rats as control. METHODS: Animal models were established using intratracheal injection of the lung and 22 107 genes were screened in the differential expression profiling of silicosis by using oligonucleotide bead array. Differential expression profiling data were analyzed by using DAVID bioinformation software. RESULTS: Totally 1567 differentially expressed genes were identified in lungs of silica exposed rats including 765 up-regulated genes and 802 down-regulated genes as compared to the normal controls. Among 406 annotated genes in KEGG pathways, 204 genes and 11 pathways were up-regulated as well as 202 genes and 3 pathways were down-regulated in silica exposed rats. CONCLUSION: All 1567 genes are involved in the formation of silicosis. The differential gene expression profile of silicosis describes the general changes in the gene expressions in silicosis at transcriptional level. Further analysis of the identified genes might help reveal the molecular mechanism of pulmonary fibrosis induced by silica.

[The clinical significance of real-time fluorescence PCR quantification of hepatocyte growth factor mRNA expression in acute leukemia].

OBJECTIVE: To detect quantitatively hepatocyte growth factor (HGF) mRNA expressions of bone marrow mononuclear cells (MNCs) in acute leukemia (AL) and investigate its clinical significance. METHODS: Total mRNA of quantitated bone marrow MNCs isolated from 67 de novo AL cases was extracted and then cDNA was synthesized. Expression of HGF mRNA was quantified absolutely using real-time fluorescence quantification PCR (FQ-PCR). RESULTS: Expressions of HGF mRNA in a group of AL were higher significantly than these in a control group (6.936 +/- 1.613, 0.407 +/- 0.170, P < 0.001), but there was similarity between a group of acute myeloid leukemia (AML) and group of acute lymphoblastic leukemia (ALL) (7.127 +/- 1.911, 6.635 +/- 0.934, P > 0.05). In AL subtypes, the expression of M5 (9.998 +/- 1.454) was higher than that of M2, M3, M4, L1, L2 and L3 (P < 0.001), but there were no differences among the latters (P > 0.05). Meanwhile, there was no statistical significance on the expressions of HGF mRNA between different age and sex (P > 0.05). In addition, expressions of HGF mRNA in the remission group were lower than these in the non-remission group (6.393 +/- 1.165, 8.041 +/- 1.848, P < 0.005). CONCLUSIONS: There are statistical significances of the expressions of bone marrow MNCs HGF mRNA among the AL group and control group. As to AL subtypes, there are no statistically significant differences between AML and ALL as well as between different age and sex. Besides, lower HGF mRNA level is correlated with better curative effect. It is suggested that HGF mRNA is a suitable index for AL diagnosis and treatment.

Study of serum argininosuccinate lyase determination for diagnosis of liver diseases.

The objectives of this research were to establish an automatic analysis method for the determination of serum argininosuccinate lyase (ASL) and to investigate the value of serum ASL test in the diagnosis of various liver disorders. According to the chemical reaction catalyzed by ASL, an enzyme-coupled reaction system was designed, and a methodology evaluation of this method was performed. A total of 291 patients with various liver diseases, 247 patients with nonliver disease and 32 healthy controls, were recruited, their serum levels of ASL and traditional hepatopathy markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and total bilirubin (TBil), were all determined, and their diagnostic values in liver diseases were analyzed and compared. Liver biopsy and the score of histopathological inflammation grading were performed in 31 patients with hepatopathy to explore the correlation between serum ASL level and hepatic histopathological change. A continuous monitoring assay method of serum ASL activity was established, which could be performed with automatic biochemistry analyzer. Methodological evaluation exhibited that the precision of this method was good indicated by the 4.0% intraassay coefficient of variation (CV), and 5.9% interassay CV. The mean recovery was 100.5%, linear range was from 0 to 167.7 U/L, and the lowest detection limit was approximately 0 U/L. All of the tested hepatopathy markers listed above were significantly increased in the liver disease group. However, levels of traditional markers of hepatopathy were all significantly increased at different degrees (all P<0.001) in patients with nonliver diseases; in contrast, there were no significantly increased ASL levels in all non-hepatopathy groups (P=0.335). The receiver operating characteristic (ROC) curve showed that the sensitivity and specificity of ASL were 100% and 91.1% (cutoff value=8 U/L), respectively, in the assessment of liver diseases. In contrast, ALT levels were 97.6% and 24.7%, and AST levels were 83.8% and 28.3% (both cutoff values=40.0 U/L), respectively. A positive correlation (r=0.417, P=0.019) was observed between serum ASL levels (86.9+/-26.5) and scores of histopathological inflammation grading (SHIG) (9.83+/-3.36). The sensitivity and specificity of ALS is much higher than that of ALT and AST for the diagnosis of liver diseases. ASL may be a more valuable marker for estimating hepatopathy.

[Construction and targeting study of eukaryotic expression vector modulated by a macrophage-specific promoter].

AIM: To construct a macrophage-specific eukaryotic expression vector and to investigate its expressing specificity. METHODS: Macrophage-specific promoter was synthesized using PCR, and substituted the CMV promoter of eukaryotic expression vector pEGFP-N1 to construct a recombinant vector (pSP-GFP), which were cotransfected with pERFP-N1 vector into different cell lines. The green fluorescent protein (GFP) and red fluorescent protein (RFP) was observed by fluorescence microscopy, and the target specificity of macrophage-specific promoter was judged by comparison of expressed level of GFP and RFP in various cell lines. RESULTS: The macrophage-specific eukaryotic expression vector was successfully constructed, which showed strong activity and specificity only in macrophage cells. CONCLUSION: The constructed pSP-GFP vector was macrophage-specific, which are potential to targeted gene therapy of intracellular bacterial infection.