STX2 Promotes Trophoblast Growth, Migration, and Invasion Through Activation of the PI3K-AKT Pathway in Preeclampsia.

OBJECTIVES: Abnormal trophoblast behaviors during pregnancy contribute to the development of preeclampsia (PE). Syntaxin2 (STX2) has been shown to be a crucial epithelial mediator in numerous diseases. However, the functions of STX2 and the mechanisms underlying its role in PE remain largely unknown. The aim of this study was to explore the role of STX2 on trophoblast biology and unravel the molecular mechanisms that contribute to the development and progression of PE. MATERIALS AND METHODS: We first compared the expression of STX2 in placental tissues from women with PE and women with normal pregnancies. Then, we investigated the role of STX2 on trophoblast proliferation, migration and invasion in HTR-8/SVneo and primary human trophoblast cells by loss or gain of function experiments. In addition, co-immunoprecipitation, pulldown and immunofluorescence assays were performed to investigate the co-localization of STX2 with other proteins, and to help clarify the mechanisms underlying STX2-mediated functions on trophoblasts. RESULTS: We demonstrated that STX2 expression was downregulated in placental tissues of women with PE compared with those from normal pregnancies. Loss and gain of function experiments further confirmed a role for STX2 in cell proliferation, migration and invasion in trophoblasts. By co-immunoprecipitation, pulldown and immunofluorescence co-localization assays, we revealed that STX2 selectively interacted with p85, a subunit of PI3K, and directly recruited p85 to the cytomembrane, thereby activating the AKT signaling pathway. We further demonstrated that the AKT activation was abolished by the use of a PI3K inhibitor (LY294002), which negatively affected STX2-mediated functions on trophoblasts. CONCLUSION: All together, our findings point to a crucial role for STX2 in PE progression. Our new insights also suggest that STX2 may be a potential diagnostic tool and a novel therapeutic target for treating PE.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis.

OBJECTIVES: To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of ß-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis. METHODS: SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of ß-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins. RESULTS: The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of ß-arrestin 2 expression in SH-SY5Y cells. CONCLUSIONS: CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the ß-arrestin 2 signal.

Analysis of Five Mushroom Toxins in Blood by UPLC-HRMS. / 血液中5ç§è˜‘è‡æ¯’ç´ çš„UPLC-HRMS分析.

OBJECTIVES: To develop a method for the simultaneous and rapid detection of five mushroom toxins (α-amanitin, phallacidin, muscimol, muscarine and psilocin) in blood by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). METHODS: The blood samples were precipitated with acetonitrile-water solution(Vacetonitril∶Vwater=3∶1) and PAX powder, then separated on ACQUITY Premier C18 column, eluted gradient. Five kinds of mushroom toxins were monitored by FullMS-ddMS2/positive ion scanning mode, and qualitative and quantitative analysis was conducted according to the accurate mass numbers of primary and secondary fragment ions. RESULTS: All the five mushroom toxins had good linearity in their linear range, with a determination coefficient (R2)≥0.99. The detection limit was 0.2-20 ng/mL. The ration limit was 0.5-50 ng/mL. The recoveries of low, medium and high additive levels were 89.6%-101.4%, the relative standard deviation was 1.7%-6.7%, the accuracy was 90.4%-101.3%, the intra-day precision was 0.6%-9.0%, the daytime precision was 1.7%-6.3%, and the matrix effect was 42.2%-129.8%. CONCLUSIONS: The method is simple, rapid, high recovery rate, and could be used for rapid and accurate qualitative screening and quantitative analysis of various mushroom toxins in biological samples at the same time, so as to provide basis for the identification of mushroom poisoning events.

Epidemiological Characteristics of Sedative-Hypnotics and Opioid Painkillers at High-Frequency Exposure. / é«˜é¢‘æš´éœ²å‚¬çœ é•‡é™è¯åŠé˜¿ç‰‡ç±»é•‡ç—›è¯çš„æµè¡Œç— å­¦ç‰¹ç‚¹.

Drug poisoning has a high incidence and serious consequences in medical institutions; its epidemiological characteristics also directly affect the changes in national laws and policies and the implementation of local management policies. Chinese statistics on drug-related abnormal death cases generally come from judicial appraisal centers and medical units. However, due to differences in work content and professional restrictions, there are differences in information management forms, which makes it difficult for appraisers to conduct a professional and systematic analysis of drug-related cases. This article focuses on the analysis of epidemiological characteristics of sedative-hypnotics and opioid painkillers and their exposure patterns in cases of poisoning death by analyzing the annual report of the American Association of Poison Control Center, combined with the characteristics of drug exposure in China.

[Inhibition of SCD1 Activity Blocks Cell Cycle Progression and Impairs Proliferation in Breast Cancer Cells].

OBJECTIVE: To investigate the expression of stearoyl-CoA desaturase-1 (SCD1) in breast cancer cell lines. To analyze the effect of inhibiting SCD1 activity on the proliferation and cell cycle of MCF-7 breast cancer cell and its mechanism. METHODS: The expression of SCD1 protein were detected by Western blot techniques in breast cancer cell lines and humanskin fibroblasts.Cell viability of MCF-7 cells treated with MF-438 was measured using MTS assay and IC50 value was calculated.The distribution of cell cycle was determined by PI staining using flow cytometry.The expression of Cyclin D1 was detected by Western blot. The expression of Akt, pAkt, pAMPK and pACC were also detected by Western blot. RESULTS: The expression level of SCD1 in MCF-7 and MDA-MB-231 cells was significantly higher than that in HSF cells (P < 0.05).MF-438 showed a significant dose-dependent proliferation inhibition effect on MCF-7 cells cultured in low serum at a concentration ranging from 100 nmol/L to 100 µmol/L with an IC50 value of (3.9±0.45) µmol/L. After intervention of 5 µmol/L MF-438 in MCF-7 cells, the proportion of cells in S phase and G2/M phase was significantly decreased (P < 0.01), the proportion of cells in G0/G1 phase increased (P < 0.01), and the expression of Cyclin D1 was significantly decreased (P < 0.05); Meanwhile, the expression of pAkt and pAkt/Akt value were significantly decreased (P < 0.05) and the expression of pAMPK and pACC levels were significantly increased (P < 0.05). CONCLUSIONS: SCD1 plays an important role in the occurrence and development of breast cancer. Inhibition of SCD1 activity can inhibit cell cycle progression and impair cell proliferation by down-regulating the Akt pathway and activating the AMPK pathway. Further research on SCD1 is expected to provide a new target for molecular targeted therapy of breast cancer.

Prostaglandin E2 restrains human Treg cell differentiation via E prostanoid receptor 2-protein kinase A signaling.

Regulatory T cells (Treg cells) belong to a class of immunosuppressive cells that control the pathological changes of autoimmunity and inflammation. Prostaglandin E2 (PGE2) is a potent lipid mediator of immune inflammation including rheumatoid arthritis (RA) that exerts its effects via four subtypes of G-protein-coupled receptors (EP1-4). The ability of PGE2 to regulate human Treg differentiation has not yet been reported. In the current study, we investigated the effects of PGE2 on the differentiation of naïve T cells from healthy and RA patients into Treg cells and the intracellular signaling involved in this process in vitro. Our data indicate that PGE2 negatively influenced the percentage of Treg cells and Foxp3 mRNA expression. The regulatory effects of PGE2 were associated with increased intracellular cAMP levels and PKA activity. EP2 receptors may mediate the inhibitory role of PGE2, since PGE2 actions were mimicked by EP2 agonist (Butaprost) and cAMP agonist (Sp-8-CPT-cAMPS) but were reversed by an EP2 antagonist (PF-04418948) and a PKA inhibitor (H-89). PGE2 negatively modulated the expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), as well as the production of interleukin (IL)-10 by Treg cells via EP2 receptors and cAMP/PKA signaling. All these findings indicate that PGE2 can inhibit Treg differentiation mediated through the EP2-cAMP/PKA signaling pathway, and suggest novel immune-based therapies for use in RA treatment.

[Effect of metformin on insulin resistance during catch-up growth in mice with fetal growth restriction].

OBJECTIVE: To study the efficacy of metformin intervention on insulin resistance during catch-up growth in mice with fetal growth restriction (FGR). METHODS: Mouse models of FGR were established by low protein diet feeding of the pregnant mice. Both the newborn female mice with FGR and normal control (NC) mice were randomized for feeding with a standard diet (SF) or a high-fat diet (HF) after weaning and treatment with gavage of either metformin or normal saline. The mice were examined for vaginal opening time and the estrous cycle at the age of 8 weeks. At the age of 12 weeks, 6 mice in anestrus from each group were fasted for 12 h for measurement of body weight, height, poundera index (PI), fasting blood glucose (FBG), fasting insulin (Fins), follicle stimulating hormone (FSH) and anti-Mullerian hormone (AMH), and the HOMA-IR was calculated. The reproductive capacity of female mice was assessed by mixing them with male mice at the ratio of 2:1. The 3 × 2 factorial analysis was conducted to determine the interactions between FGR, high-fat feeding and metformin. RESULTS: Factorial analysis showed that FGR and high-fat feeding had significant effects on the PI index, Fins, HOMA-IR, vaginal opening time, and AMH (P<0.05). Metformin significantly affected the factors related to high-fat feeding including weight, PI, FPG, Fins, HOMA-IR and estrous cycle (P<0.05) and the factors related to FGR with the exception of height and FSH (P<0.05). FGR significantly affected the factors tested except for body weight (P<0.05); high-fat feeding affected all the factors but the FSH (P<0.05); metformin affected all the factors but the height and FSH (P<0.05). In the female mice treated with saline, the pregnancy rates differed significantly between FGR mice with high-fat feeding and control mice with standard feeding, and between FGR mice with standard feeding and high-fat feeding (P<0.05). CONCLUSION: FGR mice can present with delayed puberty with rare ovulation and adulthood insulin resistance, and high-fat feeding after birth can promote the catch-up growth of FGR mice. Metformin intervention is effective for improving insulin resistance and reproductive-endocrine disorders in FGR mice during catch-up growth.

Clinical value of dual-energy spectral imaging with adaptive statistical iterative reconstruction for reducing contrast medium dose in CT portal venography: in comparison with standard 120-kVp imaging protocol.

OBJECTIVE: To evaluate the clinical value of dual-energy spectral CT with adaptive statistical iterative reconstruction (ASiR) for reducing contrast medium dose in CT portal venography (CTPV). METHODS: This prospective study was institutional review board-approved, and written informed consent was obtained from all patients. 50 patients undergoing abdominal CT were randomized to 2 groups: Group A (n = 25), using spectral CT and 350 mgI kg(-1) contrast injection protocol; Group B (n = 25), using standard 120 kVp and 500 mgI kg(-1) contrast. Spectral CT images at 60 keV and standard 120-kVp images were both reconstructed with 50% ASiR. CT number and contrast-to-noise ratio (CNR) for intrahepatic and extrahepatic portal veins were measured. The maximum intensity projection (MIP) and volume-rendering (VR) images were used for subjective evaluation. These two kinds of results were statistically analyzed. RESULTS: CNR values for the intrahepatic portal vein of the 60-keV spectral images (4.2 ± 1.1) were higher than those of 120-kVp images (3.0 ± 2.1) (p = 0.03) and were the same for the extrahepatic portal vein (5.9 ± 1.4 vs 5.9 ± 1.6, p = 0.90). The portal vein and left and right branches in the 60-keV spectral images had higher CT number and lower standard deviation than the 120-kVp images (p < 0.05). Radiation dose (dose-length product and effective dose) and subjective image quality were similar for the two groups, while the spectral CT group required 25% less iodine dose (23.1 ± 3.2 g vs 30.5 ± 5.0 g). CONCLUSION: The 60-keV spectral CT images with ASiR allow 25% reduction in the iodine dose while providing better or equal image quality as the standard 120-kVp images in portal venography with comparable radiation dose. ADVANCES IN KNOWLEDGE: Compared with conventional 120-kVp CT, the use of 60-keV spectral CT images provides 25% contrast dose reduction with similar image quality in CTPV. Compared with conventional 120-kVp CT, the use of 60-keV spectral CT images with ASiR algorithm improves CNR values for the intrahepatic portal vein.

[Nitric oxide mediated TNF-α, IL-1ß gene expression in liver induced by crush injury of rat's soft tissues].

OBJECTIVE: To explore the effect of nitric oxide (NO) on the gene expression of hepatic TNF-α and IL-1ß by crush injury of rat's soft tissues. METHODS: Rats were randomly divided into sham group, crush group, crush+aminoguanidine (AG) group, and crush+L-arginine (L-Arg) group. Activities of ALT and AST as well as NO level in serum were measured. Gene expressions of TNF-α and IL-1ß were detected with RT-PCR. RESULTS: Obvious increase in TNF-α and IL-1ß mRNA expression was detected in the crush group compared with the sham group (P<0.05). After pretreated L-Arg, expressions of TNF-α and IL-1ß mRNA were markedly increased (P<0.05). After pretreated AG, those indices obviously decreased (P<0.05). Activities of ALT and AST enhanced and NO level increased in the crush group compared with the sham group (P<0.05). Pretreatment with L-Arg or AG led to substantial increased or reduced activities of ALT and AST as well as NO levels, respectively. CONCLUSION: Endogenous NO mediated TNF-α, IL-1ß mRNA up expression in liver induced by increased production of NO after crush injury of rat's soft tissues.

Restraint stress aggravates rat kidney injury caused by a crush injury through endoplasmic reticulum stress.

BACKGROUND: The present study aimed to determine whether restraint stress aggravates kidney injury caused by a crush injury through endoplasmic reticulum stress (ERS). METHODS: In this study, Sprague-Dawley rat restraint stress, crush injury, and stressful injury models consisting of restraint stress and crush injury were established. An ERS inhibitor, Salubrinal (Sal), was administered intraperitoneally 30 minutes before induction of daily injury in the stressful injury group. At the end of the experimental procedures, plasma levels of noradrenaline and adrenaline, creatine phosphokinase, creatinine, and blood urea nitrogen were measured. Kidneys were harvested, and paraffin-embedded sections of kidney tissues were processed for hematoxylin-eosin staining and TUNEL assay to verify pathologic changes. Western blot was used to determine the protein levels of glucose-regulated protein 78, CCAAT/enhancer-binding protein-homologous protein, caspase 12, caspase 3, and MCP-1 in kidney specimens. RESULTS: Compared with crush injury, the most significant changes in kidney injury occurred in the stressful injury group, which was inhibited by Sal. The results suggested that restraint stress aggravates kidney injury caused by a crush injury, and the mechanism might involve ERS. Further study showed that double attacks induced a significant increase in the levels of glucose-regulated protein 78, CCAAT/enhancer-binding protein-homologous protein, caspase 12, and caspase 3, which was inhibited by Sal. The same changes were observed using the TUNEL assay. Double attacks also induced an increased expression of the proinflammatory cytokine, MCP-1, which was inhibited by Sal. CONCLUSION: Apoptosis and inflammation induced by ERS are important mechanisms by which restraint stress aggravates kidney injury caused by a crush injury.

Cholecystokinin receptor-1 mediates the inhibitory effects of exogenous cholecystokinin octapeptide on cellular morphine dependence.

BACKGROUND: Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the µ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed. RESULTS: Forty-eight hours after treating SH-SY5Y cells with morphine (10 µM), naloxone (10 µM) induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513) at 1-10 µM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718) did not. Interestingly, CCK-8 (0.1-1 µM), a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1-10 µM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor. CONCLUSIONS: This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.

Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis.

AIM: The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA). METHODS: Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 µmol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions. RESULTS: One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter. CONCLUSION: The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.

Cholecystokinin octapeptide inhibits immunoglobulin G1 production of lipopolysaccharide-activated B cells.

Cholecystokinin octapeptide (CCK-8) is a typical brain-gut peptide that exerts a variety of physiological actions in both the peripheral and central nervous systems. Our laboratory has previously reported that CCK-8 produces immunoregulatory action through activating CCK receptor (CCK1R/CCK2R) expression on immune cell surfaces. In the present study, we investigated the effect of CCK-8 on immunoglobulin G1 (IgG1) production in lipopolysaccharide (LPS)-activated B cells in vitro. CCK-8 inhibited the proliferation and IgG1 mRNA expression of LPS-activated B cells and therefore inhibited IgG1 production. The mechanism may be associated with the regulation of CCK-8 on transcription factors Blimp1, Pax5, Xbp1 and Bcl6. CCK-8 inhibited the expression of Blimp1, while the effect on Pax5, Xbp1 and Bcl6 varied with time, suggesting that CCK-8 acted as a complex regulator of LPS-activated B cells. The inhibitory action of CCK-8 was mainly mediated through the CCK2R pathway. These studies indicate that CCK-8 attenuates humoral immune responses and acts as endogenous immune deactivators in autoimmune diseases.

[Genetic polymorphisms of nine non-DNA combined index system short tandem repeat loci in Hebei Han population and application in paternity testing].

OBJECTIVE: To investigate the polymorphisms of 9 non-DNA combined index system (CODIS) short tandem repeats (STRs), i.e., D7S3048, D8S1132, D11S2368, D2S1772, D6S1043, D13S325, D12S391, GATA198B05, D18S1364 in Hebei Han population, and evaluate the usage of them in paternity testing. METHODS: One hundred and forty-seven unrelated healthy individuals from the Han population of Hebei province were genotyped using STRtyper10G kit including 9 STR loci on ABI 3130 Genetic Analyzer. Hardy-Weinberg equilibrium and population genetic parameters were calculated. Fourteen cases of motherless paternity testing and 2 cases of standard trios with mutation in 1 locus were detected using STRtyper10G. RESULTS: (1) Ninety-nine alleles and 336 genotypes were observed in the 9 STR loci in the population. The cumulative discrimination power(DP) was higher than 0.999,999,999. The cumulative probability of exclusion (PE) for trios and duos were 0.999,974 and 0.998,759 respectively. Departure from Hardy-Weinberg equilibrium was not observed in any of the 9 loci. (2) The combined paternity index (PI) of the 14 cases of motherless paternity testing ranged from 10³-106 for 15 STR loci in ID, whereas it reached 105-109 for 22 independent STR loci included in ID and STRtyper 10G. Possible mutation in FGA and vWA was observed in 2 cases of trios, and the combined PI was 5945 and 1840 respectively for 15 STR loci in ID. Adding STRtyper 10G to detect these 2 cases, the combined PI reached 2.76 × 107 and 4.88 × 107 respectively. CONCLUSION: The genetic polymorphism of the 9 non-CODIS STR loci included in STRtyper 10G was quite high in Chinese Hebei Han population, indicating the 9 STR loci are valuable as complement markers for ID and PP16 kit in motherless paternity testing, paternity testing with mutation and other kinds of complicated paternity testing.

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector.

AIM: To generate recombinant adenoviral vector containing calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine. METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease digestion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5' end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinantion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombinant adenoviral vector to package and amplify recombinant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expression of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting. RESULTS: The CRT-HBsAg fusion gene was characterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HBsAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 x 10(11) pfu/mL. The CRT-HBsAg fusion protein was expressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B virus gene therapy.

[Heat sensitive moxibustion for treatment of nerve root cervical spondylosis].

OBJECTIVE: To observe the therapeutic effect of heat sensitive moxibustion treatment for nerve root cervical spondylosis. METHODS: One hundred and sixty cases were randomly divided into a heat sensitive moxibustion group (n = 54), a traditional hanging moxibustion group (n = 53) and an acupuncture group (n = 53). In heat sensitive moxibustion group, heat sensitive points were explored among acupoints on neck and nucha, lateral part of forearm and crus, etc. In traditional hanging moxibustion group and acupuncture group, Jiaji (EX-B 2) points, Fengchi (GB 20), Jianwaishu (SI 14) etc. were used for hanging moxibustion and acupuncture, respectively. And scores of Pain Rating Index (PRI), as well as therapeutic effect were evaluated before and after treatment. RESULTS: The effective rate was 98.0% (50/51) in the heat sensitive moxibustion group, 83.0% (39/47) in traditional hanging moxibustion group, and 89.6% (43/48) in acupuncture group. The therapeutic effect of heat sensitive moxibustion group was better than that of acupuncture group (P < 0.05), and it was better in acupuncture group than that of traditional hanging moxibustion group (P < 0.05); PRI scores were all decreased in three groups after treatment (all P < 0.001); pain alleviation in heat sensitive moxibustion group was better than that of acupuncture group (P < 0.05), and it was better in acupuncture group than that of traditional hanging moxibustion group (P < 0.05). CONCLUSION: The therapeutic effect of heat sensitive moxibustion treatment for nerve root cervical spondylosis is better than that of traditional hanging moxibustion and acupuncture.

[Effect on mRNA and secretion levels of proinflammatory cytokines in DC infected by SARS-CoV N gene recombinant adenovirus].

OBJECTIVE: To investigate the exact mechanism of SARS-CoV pathogenesis at the protein level. METHOD: Under the condition of the establishment of dendrtic cells (DC) culture method, we used recombinant adenovirus to infect mature DC to make clear the development changes in mRNA levels and secreted protein levels of proinflammatory cytokines of IL-1beta, IL-6, IL-8 and TNF-alpha by using RT-PCR and ELISA. RESULT: We found that mRNA levels and secreted protein levels of IL-6 and TNF-alpha in DC had increased gradually after rAd-N infection during first 24 h compared with the control DC infected by rAd-LacZ (P < 0.05 or P < 0.01). CONCLUSION: It is suggested that N protein may be related to the excessive secretion of proinflammatory cytokines during SARS-CoV infection at the acute phase.

[Genetic polymorphism of four X chromosome short temden repeats loci in Hebei Han population].

OBJECTIVE: To investigate the polymorphism of loci DXS6800, DXS6797, GATA172D05, DXS986 four loci in Hebei Han population. METHODS: The genome DNA of unrelated individuals,the families and rotten materials were extracted with phenol-chloroform method and Chelex-100 method,respectively. The PCR products were detected by the polyacrylamide gel electrophoresis and DNA sequencing analysis. RESULTS: Among 150 unrelated males and 150 unrelated females from Hebei Han population, 25 alleles were found in the 4 loci. One hundred and thirty-eight haplotypes of the male were detected. The haplotype diversity reached 0.9986. CONCLUSION: The findings provided the polymorphic data of DXS6800, DXS6797, GATA172D05, and DXS986 loci in Hebei Han population. The four loci are relatively abundant in polymorphic information for identification and the obtained data of Hebei Han population can be applied to the X-STR genetic data bank.