Objective To establish a real-time fluorescent quantitative RT-PCR for detecting SARS-Coronavirus (CoV) mRNA in gargling liquid and serum of SARS patients.Methods The assay is based on simplified nested fluorescent RT-PCR. Total RNA was extracted from gargling liquid and serum by using high performance system and reverse-transcription by antisense primer.The specific TaqMan probe was designed according to the published DNA sequence of SARS-CoV polymerase and labeled with FAM and TAMRA. Standard curves for SARS-CoV quantification were prepared with serial dilutions of the recombinant plasmid.Results The method is highly sensitive and specific. The sensitivity of assay was 1?105 copies/L. The positive rate of SARS-CoV mRNA in the gargling liquid of patients was 65.0% (26/40) and 11.5% (6/52) in suspected patients, respectively. SARS-CoV mRNA was not detectable in the gargling liquid from 40 healthy individuals. The positive rate of SARS-CoV mRNA in the serum of SARS patients was 25.6% (10/39). PCR amplification products of 6 suspected patients with SARS-CoV mRNA were confirmed by sequencing and the sequence data were consistent with that of BJ01 AY278488.Conclusions Real-time fluorescent quantitative RT-PCR should be a rapid, specific tool for early diagnosis of SARS-CoV infection.

In order to investigate the possible role of ONOO- in regulatory disorder of pulmonary arterial tension in endotoxic shock, the responses of rabbit pulmonary arterial rings (PARs) preincubated with ONOO- to endothelial dependent and receptor dependent relaxants acetylcholine (ACh) and adenosine diphosphate (ADP), endothelial dependent and receptor independent relaxant A23187, endothelial independent relaxant sodium nitroprusside (SNP) and α1-adrenoceptor agonist phenylephrine (PE) were observed in vitro in accumulative manner. Results were as follow: (1) Relaxations of PARs to ACh, A23187 and ADP were markedly impaired with shift of accumulative dose response curve of each agonist to the right. Inhibition of endothelial dependent and receptor dependent or independent relaxation by ONOO- was dose dependent. (2) ONOO- incubation inhibited SNP-induced relaxation in a dose dependent manner. Accumulative dose response curve of SNP was right shift to some degree depending on the doses of ONOO-. (3) Contractile response of PARs to PE varied with the different doses of ONOO-. In PARs preincubated with 0.5 mmol/L ONOO-, contractile reponse was significantly enhanced with shift of PE accumulative dose response curve to the left, while in PARs preincubated with 1.0 mmol/L or 2.0 mmol/L ONOO-, it was markedly reduced with right shift of PE accumulative dose response curve. (4) Vehicle of ONOO- had no effect on responses to every agonist, whereas decomposed ONOO- had minimal effect on the response to PE and ADP. In contrast, relaxation of PARs to ACh, A23187 and SNP were enhanced. These results suggested that direct effect of ONOO- on pulmonary artery may be a key factor contributing to regulatory disorder of pulmonary arterial tension induced by LPS and pulmonary hypertension in the early stage of endotoxic shock.

This study was designed to invesigate vasodilatory action of exogenous peroxynitrite (ONOO-), and effect of endothelial cells on ONOO- -induced relaxation in isolated rabbit pulmonary arterial rings (PARs). Results were as follows: (1) In precontracted PARs, ONOO- could give rise to vasodilation in a dose-dependent manner. Relaxations of PARs to ONOO- at doses of 10-5 mol/L, 5×10-5 mmol/L and 10-4 mol/L were 11.09%±1.84%, 31.10%±3.53% and 64.35%±3.83%, respectively, all of which were significantly higher than those of decomposed ONOO- with 5.88%±1.27%、16.15%±1.82% and 34.44%±3.26% at same concentrations, respectively. (2) Compared with SNP and ACh, ONOO- had weak relaxant action. (3) ONOO- induced more significantly enhanced relaxation in denuded endothelial PARs than in intact endothelial PARs. (4) In this experimental condition, the relaxation of PARs to 10-6 mol/L ACh remained unchanged before and after observation of relaxation to ONOO-. (5) The relaxations of PARs to 5×10-5 mol/L ONOO- in repetitively administered manner appeared progressively decreased. These results suggested that ONOO- might be implicated in pulmonary hypertension in the early stage of endotoxic shock.

BACKGROUND:Previous studies have shown that bone marrow mesenchymal stem cels in the treatment of neurological diseases have achieved some success, which can promote neurological alterations; however, there is no breakthrough on gene and drug regulation. OBJECTIVE:To investigate the influence of ginsenosides-induced differentiation of bone marrow mesenchymal stem cels on nerve regeneration after traumatic brain injury. METHODS: A traumatic brain injury model was built in rats using hydraulic shock method, and then rat models were randomly divided into model group (traumatic brain injury group), bone marrow mesenchymal stem cel group, ginsenosides group (ginsenosides induced differentiation of bone marrow mesenchymal stem cels). At 2 weeks after transplantation, western blot assay was used to detect protein expression levels of nerve growth factor and brain-derived neurotrophic factor, immunohistochemistry assay used to detect the number of BrdU-positive cels. At 1, 3 days and 1, 2 weeks after transplantation, modified neurological severity scores were recorded. RESULTS AND CONCLUSION: The expression levels of nerve growth factor and brain-derived neurotrophic factor protein were significantly higher in the ginsenosides group than the bone marrow mesenchymal stem cel group and model group (P < 0.05). The number of BrdU positive nerve cels was also higher in the ginsenosides group than the bone marrow mesenchymal stem cel group and model group (P < 0.05). At 3 days and 1, 2 weeks after transplantation, the modified neurological severity scores in the ginsenosides group were lower than those in the bone marrow mesenchymal stem cel group and model group (P< 0.05). These findings indicate that ginsenoside-induced bone marrow mesenchymal stem cel transplantation can promote nerve regeneration in rats with traumatic brain injury, which has better outcomes than bone marrow mesenchymal stem cel transplantation alone.

BACKGROUND:Bone marrow mesenchymal stem cels (BMSCs) can promote nerve regeneration, but there are no better results because of the limitations of treatment methods. BMSC transplantation alone is not enough to achieve desired therapeutic effects. OBJECTIVE:To investigate the effect of fibroblast growth factor (FGF)-modified BMSC transplantation on functional recovery and expression of glial fibrilary acidic protein after traumatic brain injury. METHODS:Animal models of traumatic brain injury were established in Sprague-Dawley rats using hydraulic shock method, and then randomized into control group (traumatic brain injury group), BMSC group and FGF-BMSC group (FGF-modified BMSC group). After isolation and culture, BMSCs were modified by adenovirus vector-mediated FGF gene. Western blot assay was used to detect transfection efficiency and glial fibrilary acidic protein expression; immunohistochemical detection was used to detect distribution and number of BrdU positive cels in the brain; Longa score was used to evaluate the neurologic function of rats at 1, 3 days, 1, 2 weeks after transplantation; TUNEL assay was used to detect cel apoptosis in the brain. RESULTS AND CONCLUSION:Western blot results showed that FGF gene was successfuly transferred to the adenovirus vector, and capable of expressing in BMSCs; moreover, the glial fibrilary acidic protein expression of FGF-BMSC group was significantly higher than that in the other two groups (P < 0.05). The number of BrdU positive cels in the brain was significantly higher in the FGF-BMSC group than the other two groups (P < 0.05). Two weeks after transplantation, the Longa scores in the FGF-BMSC group were significantly lower than those in the other two groups (P < 0.05). TUNEL results showed that the number of apoptotic cels in the FGF-BMSC group was significantly lower than that in the other two groups (P < 0.05). These findings indicate that FGF-modified BMSCs transplantation is able to improve neurological damage after traumatic brain injury and promote neurological recovery, which is better than BMSC transplantation alone.

ObjectiveTo investigate the effects of pravastation intervention on tumor necrosis factor (TNF)-α-indueed ossifie calcification in human umbilical artery smooth muscle cells (hUASMCs). MethodshUASMCs were cultured by tissue explant in vitro, hUASMC were treated with TNF-α 50 μg/L and pravastatin of three different concentrations. The calcium deposition was determined by O-cresolphthalein eomplexone method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. ResultsPravastatin inhibited the proliferation of hUASMC (r=-0.946, P＜0.01) and decreased the cell calcium deposition (r=-0.973, P＜0.01) in a dosedependent manner. Pravastatin down-regulated the expression of BAP, OPN and BMP-2 induced by TNF-α in a dose-dependent manner (mRNA, r=-0.972, P＜0.01;BAP protein, r=-0.820, P＜0.01;OPN protein, r=-0.972, P＜0.01;BMP-2 protein, r=-0.928, P＜0.01). ConclusionPravastatin can inhibit the proliferation of hUASMC, decrease the cell calcium deposition and inhibit the ossifie calcification of hUASMC induced by TNF-α.

The morphological and molecular identifications of Cyclospora-like oocysts of dogs were underwent in the present study, in which the morphological characteristics of the Cyclospora-like oocysts firstly found in the stool samples of dogs, such as shape, size, acid-fast staining,sporulation and auto-fluorescene, were observed. According to the published sequence of the rDNA gene of Cyclospora in GenBank, 3 primers were designed and were used to amplify part of the 18S rDNA gene of dog-associated Cyclospora-like organism by nested PCR.The amplicons were purified and cloned into vector pMD19T. Then, the positive clones screened were sequenced and subjected to sequence homology and phylogenetic analysis. It was found that the morphological charactertistics of the Cyclospora-like oocysts in dogs were similar to that of the human Cyclospoa oocysts and the size of-the amplified fragment of 18S rDNA was proved to be 715 bp, that was identical to that of the target fragment. Based on the results of sequence homology and phylogenetic analysis, the dog-associated Cyclospora-like organism was identified as the Cyclospora species.

AIM：The mechanism of tumor necrosis factor-alpha (TNF-α) induced pulmonary artery hypertension(PAH) in endotoxic shock (ES) is not clear. Cholecystokinin-octapeptide (CCK-8) had anti-ES and anti-PAH effects. The impact of CCK-8 on changes in vascular reactivity and endothelial ultrastructure induced by TNF-α was studied. The role of nitric oxide (NO) was preliminarily studied. METHODS：Rabbit pulmonary artery rings were divided into four groups: TNF-α, TNF-α+CCK-8, CCK-8 and Vehicle. The rings were incubated for 2 h, 7 h or 14 h. Relaxative responses to ACh(10-8-10-5 mol/L)， SNP (10-9-10-6 mol/L) and contractile responses to PE(10-8-10-5 mol/L) were generated seperately. The NOS activity of rings was detected and the ultrastructure of endothelium was observed in the groups that incubated for 7 h.RESULTS:The relaxative responses to ACh were not affected by TNF-α and CCK-8 after incubation for 2 h. TNF-α(7 h,14 h) significantly reduced ACh-induced endothelium-dependent relaxation response of pulmonary artery. CCK-8 reversed the effect. CCK-8 itself had no effect on responses of normal pulmonary artery. Contraction to PE and relaxation to SNP were unaffected by TNF-α, CCK-8. The NOS activity increased in the TNF-α and the TNF-α+CCK-8 groups. While no significant difference was obseved between the Vehicle and the CCK-8 groups. Endothelial injury in TNF-α group and alleviated changes in TNF-α+CCK-8 group were observed. CONCLUSION:TNF-α significantly inhibits endothelium-dependent relaxation, which be one of the mechanisms of PAH induced by TNF-α during ES. It was found for the first time that CCK-8 reversed TNF-α induced impairment of endothelium-dependent relaxation and alleviated structural injury of endothelium, which might be one of the mechanisms of anti-PAH effect by CCK-8 during ES. The effects of TNF-α and CCK-8 might be related to NO.

AIM: To investigate the inhibitory effects of cholecystokinin octapeptide(CCK-8) on nuclear factor-?B(NF-?B) activities stimulated by lipopolysaccharide(LPS) by using forskolin,the activator of adenylate cyclase,and PKA inhibitor H-89 in rat pulmonary interstitial macrophages(PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-?B activities and Western blotting was used to analyze the I?B-? protein level in rat PIMs.RESULTS: The NF-?B activity was not detected in normal control rat PIMs.The NF-?B activity in LPS-treated rat PIMs was obviously higher than that in control group(P0.05).The NF-?B activity in CCK+LPS group and LPS+Fsk group were obviously lower than that in LPS group(P

AIM: To observe the changes in nitric oxide(NO) and peroxynitrite anion (ONOO - ) in the injuried lung following the ischemia-reperfusion of hind limbs and evaluate the contribution of NO and ONOO - to tissue injury. METHODS: A model of hind limbs ischemia was made by clamping infrarenal aorta with a microvascular clip and lung injury occurring after reperfusion. Lung tissue was obtained from the animals received sham operation(group 1),4 hours ischemia without reperfusion(group2), 1 hour reperfusion following 4 hours ischemia (group3) and 4 hours reperfusion following 4 hours ischemia (group4) . The contents of MDA, NO - 2/NO - 3 and the activities of SOD in the lung were examined. Immunohistochemical technique was used to determine the immunoreactivity to iNOS and nitrotyrosine(NT)-a specific "footprint" of peroxynitrite. RESULTS: Compared with group1 and group2,the contents of MDA and NO - 2/NO - 3 increased significantly (P