The expression vector of shRNA targeted to the rat angiotensin II receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in thein vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat (SHR) at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin II receptor mRNA were synthesized individually according to the sequence of the rat angiotensin II receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases, RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35.5%±3.0%, and the levels reached their lowest point after 72 h (20.7%±4% of control). At 24 and 48 h, AT1 protein was reduced to 46.9%±4.2% and 36.98%±3.7% respectively compared to control and a maximum reduction was observed after 72 h of incubation (28.1%±4% compared to controls). Plasmid-based shRNA expression systems targeted against the rat angiotensin II receptor gene were generated successfully. The shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA (siRNA) by the Dicer. Thein vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed inE. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigatedin vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMCin vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD=0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further rescarch of Id-HSP complex vaccine for B-CLL.

To explore the effect of coriaria lactone (CL)-activated astrocyte-conditioned medium on the cerebral TNF-α of normal rats, the CL-activated astrocyte-conditioned medium (ACM) was injected into the lateral ventricle of SD rats. The rats were observed for behavioral changes, and the changes of the expression of TNF-α in the cerebral cortex and hippocampus were immunohistochemically examined by employing SP method. TNF-α level was assessed by means of radioimmunoassay in homogenate of cerebral cortex and hippocampus as well as cerebrospinal fluid. Seizure episodes were observed in ACM group 30 min after the ACM injection, but they were not observed in the control group. Immunohistochemical detection showed that the immunoreaction of TNF-α in hippocampus and cerebral cortex of rats were stronger than that of the control group 4 h after the ACM injection (P<0.05). In this group, the concentrations of TNF-α in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid were higher than those of the control group (P<0.05). It is suggested that the ACM activated by CL can enhance the expression of TNF-α in normal rats, and is related to epileptogenesis.

It has been suggested that HCN₁ is primarily expressed in hippocampus, however little is known about its effects on spatial learning and memory. In the present study, we investigated the effects of non-specific HCN₁ blocker CsCl on spatial learning and memory by using Morris water maze andin situ hybridization in mice. The results showed CsCl 160 mg/kg ip for 4 days, and the mean escape latency was 34 s longer than that of normal control (P<0.01). In hippocampal tissues, staining for the HCN₁ mRNA was stronger in the DG and CA1 region of the hippocampus (P <0.05,P<0.05, when CsCl-administration group was compared with normal group). Our results suggested that CsCl could significantly affect the spatial learning and memory in mice, and HCN channel is involved in the process of learning and memory.

The differentially expressed genes between esophageal squamous cell carcinoma (ESCC) with or without lymphatic metastasis were investigated by gene chip and the lymphatic metastasis-associated genes were screened out. Expression array was used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 cases of human ESCC. The lymphatic metastasis-associated genes were screened by bioinformatics between ESCC with or without lymphatic metastasis. The results showed that 43 (4.85%) genes significantly differed between the ESCC with and without lymphatic metastasis (P<0.05), of which 18 (2.03%) were upregulated and 25 (2.82%) down-regulated. The up-regulated genes were involved in cell adhesion molecules and cell membrane receptors and the down-regulated genes were mostly cell cycle regulators and intracellular signaling molecules. It was suggested that lymphatic metastasis-associated genes were screened by gene chip, which was helpful to understand the molecular mechanism of ESCC lymphatic metastasis and lymphatic metastasis-associated genes might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.

To investigate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and calcium-activated chloride (Cl_CB) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusionin vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs under normal and chronic hypoxic condition. The effect of Cl_CB channels on PASMCs proliferation was assessed by MTT assay. The Cl_CB channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca²⁺]_i was increased. Under normoxic condition, [Ca²⁺]_i was (123.63±18.98) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (281.75±16.48) nmol/L (P<0.01). Under normoxic condition, [Ca²⁺]_i showed no significant change and no effect on Cl_CB channels was observed (P>0.05 Chronic hypoxia increased [Ca²⁺]_i which opened Cl_CB channels. The NFA and IAA-94 blocked the channels and decreased [Ca²⁺]_i from (281.75±16.48) nmol/L to (117.66±15.36) nmol/L (P<0.01). MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). Hypoxia increased [Ca²⁺]_i which opened Cl_CB channels and had a positive-feedback in [Ca²⁺]_i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Cl_CB channel may play a part in the regulation of proliferation of PASMCs.

To investigate the changes of the activity of telomerase in peripheral blood mononuclear cells (PBMCs) from senile patients with pneumonia, the telomerase activity was examined before and after the stimulation of phytohemagglutinin-M (PHA-M) in PBMCs from 10 control subjects (group A), 12 non-senile patients with pneumonia (group B) and 9 senile patients with pneumonia (group C). Also observed was the proliferative response of these PBMCs to PHA-M. The results showed that, both with or without the stimulation of PHA-M, the values of telomerase activity in PBMCs from group C patients (A values: pre-stimulation, 0.43±0.04; post-stimulation, 0.63±0.03) were significantly lower than those in PBMCs from both group A patients (A values: prestimulation, 0.65±0.05; post-stimulation, 1.26±0.13;P<0.001, respectively) and group B patients (A values: pre-stimulation, 0.63±0.03; post-stimulation, 0.93±0.03;P<0.05, respectively). The results of MTT test showed that the proliferative activity of PBMCs in group C patients (A value: 0.35±0.03) was also significantly lower than that in group A patients (A value: 0.55±0.04;P<0.05) and group B patients (A value: 0.46±0.03;P<0.05). These results indicate that the telomerase activity decreases in senile patients with pneumonia, which may be one of the mechanisms for the weakened immune function in those patients.

To investigate the protective effect of retinoic acid (RA) on hyperoxic lung injury and the role of RA as a modulator on mitogen-activated protein kinases (MAPKs), gastation 21 d Sprague-Dawley (SD) fetuses (term=22 d) were delivered by hysterotomy. Within 12–24 h of birth, premature rat pups were randomly divided into 4 groups (n=12 each): air-exposed control group (group I); hyperoxia-exposed group (group II), air-exposed plus RA group (group III), hyperoxia-exposed plus RA group (group IV). Group I, III were kept in room air, and group II, IV were placed in 85% oxygen. The pups in groups III and IV were intraperitoneally injected with RA (500 μg/kg every day). All lung tissues of premature rat pups were collected at the 4th day after birth. Terminal transferase d-UTP nick end labeling (TUNEL) staining was used for the detection of cell apoptosis. The expression of PCNA was immunohistochemically detected. Western blot analysis was employed for the determination of phosphorylated and total nonphosphorylated ERKs, JNKs or p38. Our results showed that lungs from the pups exposed to hyperoxia for 4 d exhibited TUNEL-positive nuclei increased markedly throughout the parenchyma (P<0.01), and decreased significantly after RA treatment (P<0.01). The index of PCNA-positive cells was significantly decreased (P<0.01), and was significantly increased by RA treatment (P<0.01). The air-space size was significantly enlarged, secondary crests were markedly decreased in hyperoxia-exposed animals. RA treatment improved lung air spaces and secondary crests in air-exposed pups, but had no effect on hyperoxia-exposure pups. Western blotting showed that the amounts of JNK, p38 and ERK proteins in hyperoxia-exposure or RA-treated lung tissues were same as those in untreated lung tissues (P>0.05), whereas activation of these MAPKs was markedly altered by hyperoxia and RA. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were dramatically increased (P<0.01), whereas p-JNK1/2 and p-p38 were markedly declined and p-ERK1/2 was further elevated by RA treatment (P<0.01). It is concluded that RA could decrease cell apoptosis and stimulate cell proliferation under hyperoxic condition. The protection of RA on hyperoxia-induced lung injury was related to the regulation of MAP kinase activation.

The effects of amiodarone on transmural dispersion of ventricular effective refractory periods (ERPs) in the normal and hypertrophic canine heart were investigatedin vivo. By using the programmed stimulation protocol, the ERPs of epicardium (Epi), midmyocardium (Mid) and endocardium (Endo) were measured by inserting specially-designed electrodes into the three myocardial layers before and after mainlining of amiodarone. No significant ERPs-dispersion was observed in the three layers before and after mainlining of amiodarone in the normal group. In contrast, ERPs of all the three layers were prolonged in the hypertrophic heart, while the ERPs-dispersion was reduced significantly after mainlining of amiodarone. The ERPs-dispersion was significantly increased in the hypertrophic heart but not in the normal heart using “long-short” and “short-long” interval stimulation technique. It was concluded that (1) the differences in ERPs-dispersion among the three layers were significant in hypertrophic heart, and differences were not significant in normal canine heart; (2) ERPs of each three-myocardial layers were significantly prolonged after using amiodarone, but the ERPs-dispersion decreased in hypertrophic heart and (3) the programmed extrastimulus technique of “long-short” and “short-long” intervals increased the transmural ERPs-dispersion in the hypertrophic heart.

To investigate p120 catenin mRNA expression in Non-Hodgkin's lymphoma (NHL) cell lines (U937, Raji, Jurkat and Molt4) and normal lymphocytes and explore the relationship between p120 catenin and Non-Hodgkin's lymphoma, total RNA sample was extracted by using TRIzol and reversely transcripted into cDNA. Polymerase chain reaction was performed to detect mRNA expression of p120 catenin in NHL cell lines U937, Raji, Jurkat and Molt4. Normal lymphocytes were used as control. It was found expressions of p120 catenin 1A and 3A mRNA were high in above-mentioned NHL cell lines, but neither p120 catenin 1A nor 3A was found in normal lymphocytes as shown by RT-PCR. It is concluded that both P120ctnlA and P120ctn3A mRNA transcripts were found in all NHL cell lines U937, Raji, Jurkat and Molt4 but they dont exist in normal lymphocytes, suggesting p120ctn possibly is of importance in diagnosis and therapy of lymphoma.

To investigate the protective effect of L-carnitine on myocardial ischemia-reperfusion injury in rat heart, all harvested isolated hearts were perfused on Langendorff apparatus with oxygenized K-H solution for 20 min. The hearts were then exposed to ischemia for 30 min. Following the ischemia the hearts were re-perfused with K-H solution for 120 min to serve as the control group A. Either 5 or 10 mmol/L of L-carnitine was added into the K-H solution for 20 min at the beginning of reperfusion to generate group B and group C, respectively. The derivatives of the intraventricular pressure curve (DP/DT), left ventricular developed pressure (LVDP), and coronary flux were monitored during the entire experiment. The levels of ATP, hepatin, malondialdehyde (MDA), and superoxide dismutase (SOD) in tissue, and lactic dehydrogenase (LDH), creatine phosphate kinase (CPK), malondialdehyde (MDA), and superoxide dismutase (SOD) concentration in the coronary efflux were all measured. Compared with the control group, the treatment with L-carnitine resulted in better results, i. e., higher DP/DT_max and LVDP. At the same time, ventricular fibrillation was reduced, and the levels of ATP, hepatin and SOD were all elevated. However, the concentrations of MDA, CPK and LDH were all reduced. In conclusion, L-carnitine has a protective effect on ischemia-reperfusion injury, which is partly due to its prevention of energy loss and its antioxidant activity.

The effects of fenofibrate on plasminogen activator inhibitor 1 (PAI-1) expression in human umbilical endothelial cell-derived transformed cell line-ECV 304 cells were investigated. ECV 304 cells were incubated with different concentrations of fenofibrate (0, 10, 50, 100 μmol/L) for 24 h. PAI-1 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. PAI-1 antigenic content of endothelial cells was measured by using ELISA. Fenofibrate could inhibit the PAI-1 mRNA and protein expression and reduce PAI-1 antigenic content dependently. After treatment with fenofibrate (10 μmol/L), the expression levels of PAI-1 mRNA and protein were 0.65±0.05 and 0.96±0.11 respectively, significantly lower than in the control group (0.78±0.03 and 1.21±0.15, respectively,P<0.05). PAI-1 antigenic contents (24.52±8.39) in ECV304 cells treated with 10 μmol/L fenofibrate were significantly lower than those in the control group (6.98±5.12,P<0.05). It was concluded that fenofibrate inhibited the expression of PAI-1 mRNA in ECV304 cells, and reduce the protein expression and the antigenic content of PAI-1, suggesting that fenofibrate may have an antiatherosclerotic effect on endothelial cells by PAI-1 pathway.

To observe the effects of simvastatin on nuclear factor kappaB (NF-κB)-DNA binding activity and on the expression of monocyte chemoattractant protein-1 (MCP-1) in atherosclerotic plaque in rabbits and to explore the anti-atherosclerotic properties beyond its lipid-lowering effects. Thirty-six New Zealand male rabbits were randomly divided into low-cholesterol group (LC), high-cholesterol group (HC), high-cholesterol+simvastatin group (HC+S) and then were fed for 12 weeks. At the end of the experiment, standard enzymatic assays, electrophoretic mobility shift assay (EMSA), immunohistochemical staining, and morphometry were performed to observe serum lipids, NF-κB-DNA binding activity, MCP-1 protein expression, intima thickness and plaque area of aorta respectively in all three groups. Our results showed that the serum lipids, NF-κB-DNA binding activity, expression of MCP-1 protein, intima thickness, and plaque area of aorta in the LC and HC+S groups were significantly lower than those in the HC group (P<0.05). There was no significant difference in the serum lipids between the LC and HC+S groups (P>0.05), but the NF-κB-DNA binding activity, the expression of MCP-1 protein and the intima thickness and plaque area of aorta in the HC+S group were significantly decreased as compared to the LC group (P<0.05). This study demonstrated that simvastatin could decrease atherosclerosis by inhibiting the NF-κB-DNA binding activity and by reducing the expression of MCP-1 protein.

Histone deacetylase (HDAC₁) has a high expression in many cancer cells and curcumin can inhibit the growth of cancer cells. This paper was designed to investigate the expression of HDAC₁ of Raji cells and the effect of curcumin on their proliferation and apoptosis. Raji cells were treated with 3. 125–50 μmol/L curcumin for 8–48 h and the growth inhibition rates of Raji cells were measured by MTT. The expression of HDAC₁ on Raji cells were examined by mRNA, Western blot at 24 h various concertrations (1.6–50 μmol/L). Curcumin could selectively inhibit the proliferation of Raji cells in a dose and time dependent manner, with the inhibition rate being 52.47%–82.18% (P<0.01). The up-regulation of HDAC₁ expression was observed within 24 h after the treatment with curcumin as shown by RT-PCR and Western blot. With the increase of concentration, the expression was down-regulated in a dose dependent manner. It is concluded that the expression of HDAC₁ plays an important role in the proliferation and apoptosis of Raji cells and curcumin can inhibit the growth of Raji cells at various concentrations and promote the apoptosis of Raji cells.

The eúkaryotic expression of human arresten gene and its effect on the proliferation ofiń vitro cultured vascular smooth cells (VSMCs)in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α-actin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8)in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernantants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40. 154,P<0. 01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

To investigate effect of the transplantation of mesenchymal stem cells (MSCs) in combination with nerve growth factor (NGF) on the repair of spinal cord injury (SCI) in adult rats, spinal cord of adult rats (n=32) was injured by using the modified Allen's method. One week after the injury, the injured cords were injected with Dubecco-modified Eagles medium (DMEM, Group I), MSCs (Group II), NGF (Group III), and MSCs plus NGF (Group IV). One month and two months after the injury, rats were sacrificed and their injured cord tissues were sectioned for the identification of the transplanted cells. The axonal regeneration and the differentiation of MSCs were examined by immunocytochemical staining. At the same time, rats were subjected to behavioral tests by using the open-field BBB scoring system. Immunocytochemical staining showed that axonal regeneration and the transplanted cells partially expressed neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP). At the same time, significant improvement in BBB locomotor rating scale (P<0.05) were observed in the treatment group. More importantly, further functional improvement were noted in the combined treatment group. MSCs could differentiate into neurons and astrocytes. MSCs and NGF can promote axonal regeneration and improve functional recovery. There might exist a synergistic effect between MSCs and NGF.

To explore the effect of [Ca²⁺]_i and neuronal mitochondria transmembrane potentials in hippocampus of murine cytomegalovirus (MCMV) infected mice, newborn Balb/c mice were randomly divided into two groups: a virus inoculated group and a control group. After 56 days, single cell of hippocampus was isolated, and mitochondria transmembrane potentials and the intracellular free calcium level [Ca²⁺]_i in hippocampus were measured by means of flow cytometry (FCM). Compared with the control group, the mitochondria transmembrane potentials was decreased (P<0.01) and the intracellular free calcium level [Ca²⁺]_i was increased (P<0.01) inoculated group. The dysfunction of [Ca²⁺]₁ and mitochondria transmembrane potentials in hippocampus may play an important role in the functional disorders in CMV infected CNS.

To modify the splicing pattern of Bcl-x and compare the effect of this approch with that of the antisense gene therapy in BIU-87 cell line of bladder cancer, by using 5′-Bcl-x AS to target downstream alternative 5′-Bcl-x splice site to shift splicing from Bcl-xL to Bcl-xS and 3′-Bcl-x AS AN tisense to the 3′-splice site of exon III in Bcl-x pre-mRNA to down regulation of Bcl-xL expression, the inhibitory effects on cancer cells by modification of alternative splicing and antisense gene therapy were observed and compared by microscopy, MTT Assay, RT-PCR, FACS, Westhern bloting and clone formation. The growth of cells BIU-87 was inhibited in a dose-and time-dependent maner. Its inhibitory effect began 12 h after the exposure, reaching a maximum value after 72 h. The number of cells decreased in S phase and the number increased in G₁ phase. The ability of form foci was reduced and the antisense gene therapy was approximately half as efficient as modification of alternative splicing in inducing apoptosis. It is concluded that modification of splicing pattern of Bcl-x pre-mRNA in bladder canner cell BIU-87 is better than antisense gene therapy in terms of tumor inhibition.

The therapeutic effectiveness of nutritional support in the treatment of severe chronic hepatitis and posthepatitic cirrhosis was evaluated. 143 patients with severe chronic hepatitis and 83 with posthepatitic cirrhosis were evaluated with SGA for assessing the nutritional status before the treatment. Patients with severe chronic hepatitis were divided into three groups: group A subject to enteral nutrition (EN) and parenteral nutrition (PN), group B subject to comprehensive treatment (CT)+PN: group C subject to CT+EN. The patients with posthepatitic cirrhosis were divided into two groups: group D receiving CT and group E receiving CT+PN+EN. The function of liver and kidney and nutritional status were monitored to asses the therapy in 6 weeks. The results showed before treatment, over 90% patients had moderate to severe malnutrition. After nutritional support, the liver function (ALT, T-bil) and nutritional status (TP, TC) in group A was improved significantly as compared with that in groups B andC (P<0.05). Compared group D, the values of TP and Alb were increased significantly in group E (P<0.05), but the levels of ALT, AST and T-bil had no obvious change. It was suggested that most patients with severe chronic hepatitis or posthepatitic cirrhosis had malnutrition to varying degrees. The nutritional support treatment could obviously improve the nutritional status of these patients, and was helpful to ameliorate the liver function of the patients with severe chronic hepatitis. Among the methods of nutritional support treatment, PN combined with EN had the best effectiveness.

To investigate the features of various hepatitis virus infection in intravenous drug users (IVDU), we conducted an epidemiological survey of hepatitis viruses including hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis G virus (HGV) in IVDU. The correlation of T_H lymphocyte cytokine and hepatitis virus infection was examined. A study population of 406 IVDU consisted of 383 males and 23 females. HBV-DNA and HCV-RNA were detected by fluorescence quantitative polymerase chain reaction. HBsAg, HBeAg, anti-HBc, anti-HCV, HDV-Ag and anti-HGV were assayed by ELISA. The levels of cytokines of T_H1 and T_H2 were measured by ELISA. The similar indices taken from 102 hcalthy persons served as controls. The infection rate of cach virus among IVDU was 36. 45% for HBV, 69. 7% for HCV, 2.22% for HDV, and 1.97% for HGV, respectively. The co-infection rate of HBV and HCV was detected in 113 of 406 (27. 83%). In contrast, among controls. the infection rate was 17.65% for HBV and 0% for the other hepatitis viruses. The levels of PHA-induced cytokines (IFN-γ and IL-4) and the level of serum IL-2 were obviously decreased in IVDU. On the other hand, the level of serum IL-4 was increased. The IFN-γ level was continuously decreased when the IVDU was infected with HBV/HCV. In conclusion, HBV and HCV infection were common in this population of IVDU and they had led to a high incidence of impaired T_H1 cytokine levels.

In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encodingHA andHA₁ of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells.HA andHA₁ genes were amplified by RT-PCR and cloned into pcDNA3. 1(+) to generate pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA₁, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA₁ were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA₁. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA₁, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

In order to study whether patients with schizophrenia have cerebral injury, neuron-specific enolase (NSE) and myelin basic protein (MBP) in cerebrospinal fluid (CSF) of 33 patients with first episode schizophrenia and 9 from the control group were determined by double antibody sandwich enzyme immunoassay method. The results showed that there was significant difference in the NSE contents between the experimental group and control group (P<0.01). The NSE contents in CSF in the experimental group were positively correlated with MBP in schizophrenia patients (P<0.05). These findings suggested that patients with schizophrenia had cerebral injury.

To examine the aging related changes of microglia and astrocytes in hypothalamus of rats after intraperitoneal injection of hypertonic saline in rats, old- and young-aged rats were injected with hypertonic saline solution into peritoneal cavity. Lectin histochemical techniques using Ricinus communis agglutinin-1 (RCA-1) and immunocytochemical method employing antibody against glial fibrillary acidic protein (GFAP) were used to demonstrate microglia and astrocytes in the hypothalamus of the rats, and the positively-stained cells were analyzed by computer-assisted image analysis system. Our results showed that the numbers of microglia and astrocytes were significantly increased in the hypothalamus of old-aged rats. After intraperitoneal injection of hypertonic saline, the number of microglia was significantly decreased in the hypothalamus of both young- and old-aged groups. After introperitoneal injection of hypertonic saline, the number of GFAP positive cells was significantly increased in the hypothalamus of young rats, but the number of GFAP positive cells did not show significant change in the hypothalamus of old rats. It is concluded that in the hypothalamus of old-aged rats, the increase of microglia may be related with the aging or degeneration of neurons, and the increase of astrocytes may provide more nourishment required by the aged neurons. The microglia and astrocytes in the hypothalamus of the two group rats may be affected by hypertonic saline, and the response of these cells to the stimuli is characterized by some aging-related changes.

To investigate the neuroprotective effect of melatonin (MT) on retinal ganglion cells (RGCs) in rats with ischemia reperfusion injury (RIR), 24 healthy SD rats were randomly divided into two groups: group A and group B. RIR model was induced in the left eyes by increasing the pressure of the anterior chamber. Group A was treated with 10% alcohol-normal saline (1 mL/kg/d, ip), while group B was treated with 0.5% MT (1 mL/kg/d, ip). On the basis of the time interval between the left eyes RIR and the sacrifice, rats in both group A and group B were further divided into 3 subgroups: groups A1 and B1 (days 7), groups A2 and B2 (days 14), groups A3 and B3 (days 30), with 4 rats in each subgroup. 7 day before the sacrifice, 3% fluorogold was bilaterally injected into superior colliculi and geniculate body. The eyes were enucleated after being sacrificed, and mounting of the retina from both eyes was performed on a slide and observed under a fluorescence microscope. Four photos were taken from each of the four quadrants of the retina. The labeled-RGCs were counted by using a computerized image analyzer. The rate of the labeled-RGCs was used for statistical analysis. Our results showed that, in group A, the rate of the labeled-RGCs was (77.16±6.35) %, (65.53±7.01) %, (53.85±4.38) % on day 7, 14 and 30. In group B, the rate of the labeled-RGCs was (81.33±9.27) %, (79.80±8.36) %, (80.34±11.05) % on day 7, 14 and 30. In group B, which was treated with MT after RIR, the rate of labeled-RGCs was significantly higher than that of group A on day 14 and day 30 (P<0.05). It is concluded that, in the RIR rats, MT therapy could increase the survival rate of the RGCs and could rescue and restore the injured RGCs.

To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on the proliferation and PCNA (proliferating cell nuclear antigen) expression of cultured human retinal pigment epithelium cells, human retinal pigment epithelium cells (RPE) were cultured from normal adults who died accidentally. The effects of PDTC on the proliferation of RPE cells were examined by using methyl thiazlyl tetrazolium (MTT) assay. The effects of PDTC on the PCNA expression of RPE cells were immunohistochemically examined by employing biological image analysis system (BIAS). After treatment with PDTC of various of concentration ranging from 0.062 to 1 g/L for 24 h, or concentrations ranging from 0.031 to 1 g/L, the proliferation of RPE cells decreased in a dose-dependent manner. After treatment with PDTC of concentration varying from 0.062 to 1 g/L for 24 h, the PCNA expression was also suppressed in a dose-dependent manner. It is concluded that PDTC can inhibit the proliferation of RPE cellsin vitro in a dose-and time-dependent manner, at least in part, by down-regulating the expression of PCNA. PDTC may be used to prevent and treat the proliferative vitreoretinopathy (PVR).

To study corneal lymphangiogenesis after corneal transplantation, corneal allogenic transplantation models were established in rats. 8 female Wister rats were used as donors, and 16 Sprague Dawley (SD) rats were used as recipients and 2 SD served as controls. Corneal lymphangiogenesis and hemangiogenesis was examined by electron microscopy 1 and 2 weeks after corneal penetrating transplantation, and the expression of lymphatic vessel endothelial receptor (LYVE-1) was examined 1, 3, 7, 14 days after the transplantation respectively. In addition, 19 allograft failed human corneas were examined by 5′-nase-alkaline phosphatase (5′-NA-ALP) double-enzyme-histochemistry staining to detect corneal lymphangiogenesis and hemangiogenesis. By immunohistochemistry for LYVE-1, it was found that blown lymphatics were localized in the stroma 3 days after the corneal transplantation. With electron microscopy, new lymphatic vessels and blood vessels were found 1 and 2 weeks after the corneal transplantation. By 5′-NA-ALP enzyme-histochemistry, corneal hemangiogenesis was found in all allograft failed human corneas and 5 of 19 (26. 3%) cases had developed corneal lymphangiogenesis. It is concluded that corneal lymphangiogenesis is present after corneal transplantation, which may play an important role in allograft rejection.

To investigate the expression of vaginal IL-23 and its role in experimental murine vaginal candidiasis and its relationship with infection and immune status, immuno-competent (group A) and immuno-suppressed (group B) murine models of vaginal candidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls (group C). The level of IL-23 p19 mRNA in murine vaginal tissue was determined by RT-PCR. Significantly increased levels of IL-23p19mRNA were observed on the 4th, the 7th and 14th day after inoculation in immuno-competent group when compared with that in control group (P<0.01,P<0.05), However, significant increase of IL-23 p19mRNA were only observed on the 7th day and the 14th day after inoculatuon in immuno-suppressed groups (P<0.05). On the 4th and 7th day, the levels of Il-23 p19mRNA were significantly increased in immuno-competent group, than those in immuno-suppressed group (P<0.05). Local IL-23 may play a role in the pathogenesis of murine vaginal candidiasis and has a protective function during infection. Low vaginal IL-23 level may correlate with the increased susceptibility toCandida albicans in immuno-suppressed group.

To study the relationship between mutation of the inverted repeat sequence (IR) in the multiple transferable resistant system (mtr) of Neisseria gonorrhoeae (NG) and its multiple antibiotic resistance, minimal inhibitory concentrations (MICs) for the clinically isolated strains were tested by agar-dilution-method. The mtr systems IR gene of NG was sequenced after amplification by polymerase chain reaction (PCR). Either two susceptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a single-base deletion (A/T). The result suggests that a single-base deletion of the thirteen-base IR sequence in mtr system of NG might result in multiple antibiotic resistance but is not associated with single antibiotic resistance.

To explore the feasibility of using tissue Doppler imaging technique for the evaluation of fetus's left ventricular diastolic function, and to confirm its reliability by comparing it with traditional methods, this study examined 61 pregnant women in whom satisfactory images were obtained of fetal echocardiography. The peak velocity of blood stream were measured, including E, A and E/A at mitral valve orifice on the four chamber view with pulse wave. And then tissue Doppler imaging mode was employed to measure the velocity of mitral valve annulus including Ea, Aa, Sa and Ea/Aa. Correlation analysis was conducted between the velocity of orifice and that of annulus in terms of gestation age. And then correlation analysis was performed between above data and gestation ages. A positive correlation was found between the velocity of orifice and that of annulus, and the velocity increased with the gestation age. The change was the most significant between the 28th and the 34th week of gestation age. Our study showed that it is feasible to evaluate the fetus's left ventricular diastole function by tissue Doppler imaging. Its stability can avoid the influence of fetal heart rates and preload.

To explore the application of potassium titanyl phosphate (KTP) laser delivered via bronchofiberscope in the treatment of endobronchial tuberculosis. 36 patients with a diagnosis of endobronchial tuberculosis, with age ranging from 15 to 40 y were treated with KTP, laser between Dec. 2002 and July 2004 (designated as treatment group). The other 36 patients diagnosed as having endobronchial tuberculosis (aged 18 to 42 y, with a mean age of 33.5 y) without having received KTP laser treatment were included in a control group. Our results showed that the effective rates, in terms of recovery of bronchial lumen and cleanup of caseous necrotic mass were significantly higher in the treatment group 8 weeks after the treatment (P<0.01), and the healing rates of atelectasis and obstructive infection were also significantly higher in the treatment group (P<0.05 andP<0.01), but the incidence of complication after 8 weeks was no significant difference (P<0.05). No significant changes were found in SaO₂ and HR before, during and after the operation in the treatment group (P>0.05). It is concluded that KTP laser is an effective therapy for endobronchial tuberculosis.

To provide a scientific basis for determining the health surveillance period of dust-exposed workers, data of a retrospective cohort study was re-analyzed with emphasis on natural course of silicosis. 33640 workers exposed to silica dust who were employed for at least 1 year from 1972 to 1974 in twenty Chinese mines or pottery factories were included as subjects, and were followed up till December 31, 1994. The cohort included subjects from 8 tungsten mines, 4 tin mines and 8 pottery factories. Our results showed that the mean latency of silicosis, for all the cases of the cohorts, was 22.9±9.8 y. 52. % of silicosis was diagnosed approximately 9.1±5.7 y after the dust exposure had ceased. The progression rates of silicosis from stage I to II and from stage II to III were 48.2% and 18.5%, and the duration was 4.1±0.2 and 6.8±0.2 y, respectively. The survival times of silicosis stage I, II and III, from the year of diagnosis to death, were 21.5, 15.8 and 6.8 years, respectively. There was 25% of the silicosis patients whose survival time was beyond 33 y. The mean death age of all silicosis cases was 56.0 y. The death age increased to 65.6 y in the middle of 1990s. Among dust-exposed workers, subjects who became suspected case (0⁺) accounted for 15.0%. 48.7% of the suspected silicosis cases developed to silicosis, and the average year from the time of being suspected of the disease of the first stage of silicosis was 5.1 y. The natural characteristics, as mentioned above, varied with different mines and factories. We are led to conclude that silicosis is chronic in nature, but progress quickly. As a serious occupational disease it significantly reduces the life span of exposed workers. The study of its natural history is of importance for the development of health surveillance criteria for dust-exposed workers.

To find a component with the strongest anti-endotoxin effect from the four components (F₀₂₁, F₀₂₂, F₀₂₃, and F₀₂₄) of chloroform extract (F₀₂) ofRadix Isatidis, the protective effects of the five components on endotoxin-challenged mice and their effects on the production of TNF_α and IL-6 by macrophages from the endotoxin-challenged mice were compared. It was found that the five components all had protective effects on endotoxin-challenged mice in terms of mortality. The mortalities of the mice challenged by 2.42 mg/kg endotoxin were 30%, 50%, 20%, 50% and 60% after treatment with F₀₂, F₀₂₂, F₀₂₃, and F₀₂₄. The 5 components all had inhibotory effects on the production of TNF_α and IL-6 by macrophages from the mice. At a concentration of LPS of 50 ng/mL, the inhibitory rates of F₀₂, F₀₂₁1, F₀₂₂, F₀₂₃, and F₀₂₄ for the production of TNF_α and IL-6 were 76.54%, 30.86%, 78.60%, 36.63%, 2.06% and 75.85%, 18.45%, 77.68%, 24.41%, 3.64% and at the dose of 100 ng/mL, the inhibitory rates were 49.65%, 16.86%, 66.97%, 13.39%, 7.16% and 59.78%, 1.28%, 61.86%, 2.08%, 1.44%, respectively. It is concluded that the component F₀₂₂ ofRadix Isatidis has the strongest anti-endotoxin effect.

Protective effect and mechanism of electroacupuncture (EA) on acute reperfusion ventricular arrhthmia was investigated. Ventricular arrhythmia was induced by occlusion of the proximal left anterior descend (LAD) branch of coronary artery for 5 min and followed with 15 min reperfusion. EA on acupoint “Neiguan”, “Jianshi” was performed at 30 min before ligation and continued another 5 min during ischemia. Isoprenaline (20, 30 and 50 μg/kg) or atropine (1 mg/kg) was intravenously injected at 5min before ischemia. The results showed that EA significantly decreased the incidence of ischemia/reperfusion (I/R) induced ventricular tachycardia (VT), ventricular fibrillation (VF) and mortality as compared to I/R group. Atropine partially suppressed the EAs effect of antiarrhythmia; Isoprenaline increased the incidence and severity of reperfusion arrhythmia, which was inhibited by EA, but this inhibition of EA was blocked with increasing dose of isoprenaline. The results indicated that EA treatment could prevent the occurrence of reperfusion ventricular arrhythmia in rats with myocardial ischemia, and its mechanism might be related to the regulation of EA on the β-adrenoceptors and M-cholinergic receptor activation in myocardium.