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Objective To evaluate comprehensive therapy in the treatment of keloid effect and the patients' satisfaction.Methods A total of 523 patients with comprehensive treatment,according to the treatment,were divided into group A (surgery combined radiotherapy) and group B (surgery plus corticosteroids),group C (hyperbaric oxygen in combination with radiotherapy),and the therapeutic effect of patients with satisfaction was analyzed.Results Total effective rate of three groups of patients were 69.47％,89.13％,90.32％,respectively.Effective rate in group A was higher than that of group B and group C,there was statistically significant difference between groups A and C (P ＜ 0.05),but no significant difference between the group A and group B (P＞0.05).Difference was statistically significant between three groups of patients' satisfaction,group B better than group A and group C,the difference between group B and C group was statistically significant (P ＜ 0.05).Conclusions Effectiveness of the three combined therapies is obvious,in which radiotherapy plus hyperbaric oxygen is most effective.
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Objective Detecting plasma level of circular RNA(circRNA)hsa_circ_0009024 in pulmonary tuberculosis(TB)patients,and evaluating its diagnostic value for TB.Methods From January 2016 to December 2016, a hosptial-based, case-control study was performed, which include 90 untreated active pulmonary tuberculosis patients(TB group),75 healthy people(healthy control)and 84 patient with other diseases(other disease group).Plasma level of circRNA hsa_circ_0009024 was detected by real-time quantitative polymerase chain reaction.Furthermore, the 90 patients with TB were divided into different subgroups according to cavity formation and the lung fields involvement: patients without lung cavity(55 cases)vs those with lung cavity(35 cases),patients with involvement of <2 lung fields(49 cases)vs≥2 lung fields(41 cases).Plasma levels of hsa _circ_0009024 of 41 TB patientswere monitored andcomparedbefore and after 3 months anti-TB therapy.The sensitivity and specificity of plasma hsa_circ_0009024 were analyzed by using the receiver operating characteristic(ROC)curve.The comparison between two groups was performed with Mann-Whitney U test and the comparison among multigroupswas conducted with Kruskal-Wallis H test.Results Plasma levels of hsa_circ_0009024 in TB patients[1.98(1.42, 2.71)]were significantly higher than healthy controls[1.03(0.78,1.33)]and other disease groups[1. 13(0.77,1.51)](H=76.58,P<0.0001).Plasma levels of hsa_circ_0009024 in cavity pulmonary TB patients were higher than pulmonary TB patients without cavity(U=392.50,P<0.0001).Plasma levels of hsa_circ_0009024 in TB patients with involvement of ≥2 lung fields were higher than <2 lung fields(U=590.50,P=0.0008).As compared to pre -treatment[2.01(1.41, 2.71)], the plasma hsa_circ_0009024 levels decreased significantly in 3 months[1.22(0.85,1.47)](U=292.50,P<0.0001)after anti-TB therapy.The area under the receiver operating characteristics curve(AUC)of plasma hsa_circ_0009024 in discriminating the patients with TB from normal controls, pneumonia patients and lung cancer patients were 0.841 and 0.811, respectively.Conclusion The hsa_circ_0009024 can be used as a potential biomarker in TB diagnosis and monitoring.
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Objective: To study the relationship between endoplasmic reticulum stress (ERS) and adiponectin;to explore the role of ERS for increasing myocardial ischemic vulnerability in type 2 diabetes mellitus (DM) mice. Methods: Type 2 DM model was established by high fat diet with streptozotocin (STZ) injection. A total of 35 C57BL/6J male type 2 DM mice were divided into 4 groups: ①Control group, n=5. ②Tauroursodeoxycholic acid (TUDCA) group,③Thapsigargin (TG) group and ④Normal saline group. The mice in Groups ②, ③, ④were fed by high fat and high glucose diet by injecting streptozotocin (STZ), in the last 3 weeks and respectively received intraperitoneal injections of TUDCA (250 mg/kg), thapsigargin (TG) (300μg/kg) and normal saline twice a day, n=10 in each group. Then myocardial infarction (MI) model was established in 5 mice from each group. 72 hours later, the MI ranges were measured, serum levels of adiponectin were detected, mRNA expressions of adiponectin and CHOP in myocardial tissue were examined. Results: The MI range in TUDCA group (21.47 ± 2.85)%and in Normal saline group (39.92 ± 4.28)%were both lower than TG group (66.56 ± 8.15)%, both P Conclusion: ESR could increase myocardial vulnerability in type 2 DM mice which might be related to down-regulating adiponectin expression.
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Objective To investigate the effects of lincRNA-cox2 on the polarization of murine RAW264. 7 macrophages by analyzing the expression of lincRNA-cox2 in RAW264. 7 macrophages of M1 and M2 phenotypes. Methods Murine RAW264. 7 cells were induced by IFN-γand LPS to polarize to M1 phenotype, and were induced by IL-4 to polarize to M2 phenotype. The expression of lincRNA-cox2 in M1 and M2 macrophages were analyzed by real-time quantitative PCR ( RT-PCR) . We designed and synthesized siRNA oligo for lincRNA-cox2 and unrelated sequences. Then the siRNA oligo and NC oligo were transfected into RAW264. 7 cells by LipofectmineTM 2000. The transfected RAW264. 7 cells were induced by IFN-γand LPS or by IL-4 to polarize to M1 or M2 macrophages. Enzyme linked immunosorbent assay ( ELISA) was performed to measure the secretion of IL-10 and IL-12 induced in different conditions. The expression of in-ducible nitric oxide synthase ( iNOS ) , TNF-α, arginase 1 ( Arg-1 ) and found in inflammatory zone 1 (Fizz1) at mRNA level were detected by RT-PCR. The M1 macrophages were transfected with siRNAs to knock down the expression of lincRNA-cox2 for analyzing the biological effects of lincRNA-cox2 on the polar-ization of macrophages. Results The relative expression of lincRNA-cox2 in M1 macrophages was signifi-cantly higher than that in RAW264. 7 cells and M2 macrophages. Compared with the control group, the RAW264. 7 cells transfected with lincRNA-cox2-siRNA showed decreased secretion of IL-12 and inhibited expression of iNOS and TNF-αat mRNA level after IFN-γand LPS induction, but increased secretion of IL-10 and enhanced expression of Arg1 and Fizz1 at mRNA level after IL-4 induction. Transfecting the M1 mac-rophages with lincRNA-cox2-siRNA inhibited the secretion of IL-12, but promoted the secretion of IL-10. Conclusion This study indicated that lincRNA-cox2 was involved in the regulation of macrophage pheno-types by promoting the polarization to M1 macrophages and inhibiting the polarization to M2 macrophages.
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Protein disulfide isomerase (PDI) is one of thiol-disulfide oxidoreductases that mainly located in the endo?plasmic reticulum (ER). It is generally known that PDI caralyzes the formation,rearrangement,breakage of disulfide bonds, and this enzyme is effective in regulation of protein folding. Now it is also known as a biomarker of cardiovascular disease. Protein disulfide isomerase can reduce infarct size and myocardial apoptosis in acute myocardial infarction (AMI). PDI can also improve changes of cardiac vulnerability in diabetic cardiomyopathy (DCM). Further more, it is also shown that PDI play an important role in hypertension and thrombosis. Therefore, this paper review the effects of protein disulfide isomerase in cardiovascular diseases.
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Objective To construct a lentiviral vector-based short hairpin RNA (shRNA) targeting the gene encoding tryptophan-aspartate containing coat protein ( TACO) and to evaluate its inhibitory effect on the expression of TACO , and to further elucidate its effects on the phagocytosing and intracellular killing of My-cobacterium tuberculosis (M.tb) by macrophages and the possible mechanisms.Methods Three shRNA frag-ments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentivi -ral vector pSicoR .The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells.Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW 264.7 cells transfected with the concentrated lentivirus .The most effective lentivirus was screened out to transfect the RAW 264.7 cells for 48 hours, followed by infection those cells with M.tb strains.The entry and intracellular survival of M .tb strains in RAW264.7 cells were de-termined by bacterial culture at indicated time points .The colocalization of M .tb and lysosomes was detected by immunofluorescence staining .The cyto-ID autophagy kit was used to detect the cellular autophagy and the auto-phagy-associated protein LC 3 was determined by Western blot assay .Results The recombinant lentiviral vec-tors were successfully constructed and confirmed by sequencing analysis .Decreased expression of TACO in RAW264 .7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours.The most effective lentivirus , LV-pSRT1, decreased the expression of TACO by 85.24%and 69.00%at the mRNA and protein levels, respectively.The bacterial loads in LV-pSRT1 trans-fected RAW264.7 cells were significantly decreased at the time point of 0 h after M.tb infection as compared with those in the control lentivirus treated cells (5.50×104 vs 8.1 ×104, P<0.05).Compared with the RAW264 .7 cells transfected with control lentivirus , the survival rate of intracellular M .tb strains in LV-pSRT1 transfected cells was significantly decreased at the time point of 48 h (134.54% vs 213.58%, P<0.05) and 72 h (148.18%vs 262.96%, P<0.05) considering the bacterial load at the time point of 0 h as the standard. The immunofluorescence staining demonstrated that the colocalization of M .tb strains with lysosomes was signifi-cantly enhanced in LV-pSRT1 transfected cells as compared with that in control lentivirus treated cells (75.67%vs 10.66%, P<0.05).Moreover, significantly enhanced autophagy and relative expression of LC 3Ⅱ protein were observed in RAW264.7 cells with TACO gene knockdown (16.20%vs 8.50%, P<0.05;0.51 vs 0.34, P<0.05).Conclusion The lentiviral vector-based shRNA targeting TACO gene could effectively knockdown the expression of TACO protein , decrease the entry and increase the intracellular killing of M .tb strains in mac-rophages.The enhanced intracellular killing of M .tb strains by macrophages was associated with the increased fusion of M.tb-containing phagosome and lysosome .
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Objective To construct the secretory expression vector of recombinant human C-reactive protein(rhCRP) for its se-cretory expression in Pichia pastoris ,rhCRP was expressed as a secretory protein and purified ,and the immunity reactivity of the purified protein was identified .Methods The DNA fragment of rhCRP which was designed and synthesized was cloned into pPICZαA vector .Recombinant plasmid pPICZαA/rhCRP was linearized by SacⅠand transformed into Pichia pastoris X-33 by elec-trotransformation .The rhCRP was secreted into the medium under the methanol induction .RhCRP was purified by Histamine affin-ity chromatography .The purified rhCRP was identified by SDS-PAGE and Western blotting ,and its immunity reactivity and stabili-ty was identified by indirect ELISA .Results The pPICZαA/rhCRP expression vector was successfully constructed .The rhCRP of 23 × 103 was inducted and successfully expressed as a secretory protein by the recombinant Pichia pastoris strains .The rhCRP was purified by one step up to 90 .42% purity ,and it was showed good immunity and stability by indirect ELISA .Conclusion The rh-CRP with higher purity and immunoreactivity was successfully obtained by using the Pichia pastoris expression system ,which pro-vided an important experimental basis for producing anti-human CRP antibodies and developing testing CRP reagent .
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Objective To investigate the diagnostic value of microscopic observation drug susceptibility assay (MODS) in extrapulmonary tuberculosis. Methods MODS technology was constructed by using 24-well cell culture plate and liquid culture.Ziehl-Neelsen smear,Lowenstein-Jensen culture and MODS were used to detect Mycobacterium tuberculosis in 74 pleural fluid samples collected from patients with tuberculous pleurisy,63 cerebrospinal fluid samples collected from patients with tuberculous meningitis and 18 samples collected from non-tuberculosis suspects.The immunochromatography was used to distinguish Mycobacterium tuberculosis from nontuberculosis mycobacteria. The results of Ziehl-Neelsen smear, Lowenstein-Jensen culture and MODS were compared by x2 test.Results The positive rates of MODS,Lowenstein-Jensen culture and Ziehl-Neelsen smear were 58.1 ％ (43/74),18.9 ％ (14/74 ) and 6.8％ (5/74),respectively in tuberculous pleurisy patients; 54.0％(34/63),20.6％ (13/63) and 4.8％ (3/63),respectively in tuberculous meningitis patients.The positive rate of MODS technology was significantly higher than that of Lowenstein Jensen culture in tuberculous pleurisy patients (x2 =24.00,P＜0.01) and tuberculous meningitis patients (x2 =14.97,P ＜ 0.01). Each Mycobacterium obtained from MODS and Lowenstein-Jensen culture was identified as Mycobacterium tuberculosis by immunochromatography.All of the 18 pleural fluid and cerebrospinal fluid samples which collected from non-tuberculosis suspects were all negative detected by Ziehl-Neelsen smear,Lowenstein-Jensen culture and MODS.The median time to culture positive of MODS was 9 days in cerebrospinal fluid and 14 days in pleural fluid samples,which were both significantly shorter than that of Lowenstein-Jensen culture (31 days in both cerebrospinal fluid and pleural fluid samples). Conclusion Compared to conventional microbiological diagnosis methods,MODS is a rapid detection method of Mycobacterium tuberculosis with a higher positive detection rate,which is suitable for rapid diagnosis of extrapulmonary tuberculosis.
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Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.
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Objective To determine the level of toll-like receptor4 (TLR4) in the kidney of rats with the Multiple Organ Dysfunction Syndrome (MODS),and to investigate the early phase of kidney damage in MODS.Methods The experiment was done at experimental center of medical college of Three Gorges University.Forty Adult male Sprague-Dawley (SD) rats were randomly (random number) divided into two groups,namely the normal control group and the MODS model group.The rats of model group were further divided into four sub-groups as per different intervals (6 h,12 h,24 h and 48 h),and there were 8 rats in each groups.The animal models of MODS were established by two hits,the left eyeball of each model rat was removed to bleed to 2 mL/100 g,and four hours later,Lipopolysaccharide ( LPS,5 mg/kg) was injected into intraperitoneal cavity of model rats.The same volume of saline was injected intraperitoneally into rats of control group.All rats were sacrificed at various intervals.The histological changes in the kidney tissue were observed by naked eye and under light microscope.The lever of TLR4 proteins in serum and kidney tissue were detected by using flow cytometry (FCM).One-way ANOVA was used for comparison among multiple groups.Results (1) There were no histopatholagical changes in kidney of rats in control group,and the kidney injury was serious in rats with MODS.(2) Compared with the rots of control group,the level of TLR4 in kidney tissue of rats with MODS increased at 6 hours and reached peak 12 hours later ( P ＜ 0.01 ),and then decreased 24 hours ( P ＜ 0.01 ).There was no significant difference in levd of TLR4 between two groups 6 and 48 hours.Compared with the rats of control group,the level of TLR4 in peripheral blood leucocyte of rats with MODS increased significantly 6 ～ 48 hours after LPS ( P ＜ 0.01 ).The concentrations of serum and kidney tissue TLR4 proteins were positively correlated with each other ( r =0.893,P ＜ 0.05 ).Conclusions The level of TLR4 markedly increased in the kidney tissue at early stage of MODS,and the TLR4 may play an important role in the pathogenesis of kidney injury in MODS.
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Objective To explore the clinical therapeutic effects of intravenous infusion of dihiazem in com-bination with metoprolol for treating aortic dissection. Method From June 2005 to January 2008, fourteen patients with aortic dissection (male 8, female 4) in the First Hospital of Yichang,were treated with diltiazen 1～5 μg/(kgrate 30 min,60 min, 120 min,6 h, 1 d and 7 d after treatment were recorded. All data were analyzed using self-matching t test. Results The heart rate reduced significantly 60 min after treatment. The heart rate reduced (21±5) beats/min from the baseline. The total effective rate was 100% .The blood pressure reduced significantly 30min after treatment. The systolic pressure reduced to (126.2±11.1 ) mmHg and diastolic pressure declined to (80.3±8.1) mmHg. No severe cardiac side-effect observed. Conclusions Dihiazem in combination with meto-prolol can reduce heart rate and blood pressure markedly and safely in aortic dissection patients.
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Objective To construct eukaryotic expression recombinant plasmid containing human granulysin(GLS) and investigate the effect of GLS expression in macrophage RAW264.7 cells on the bactericidal activity against intracellular Mycobacterium tuberculosis.Methods GLS gene was amplified by nested-polymerase chain reaction(PCR) from human cytotoxicity T lymphocyte(CTL) activated by allogenic antigen,and inserted into pBudCE4.1 vector to construct recombinant plasmid.Subsequently,the plasmid was transfccted into RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The expression of GLS was detected by nested-PCR and immunocytochemistry method.The RAW264.7 cells were lysed after transfected for 96 h,then acidfast stained,cultivated and colony count were done to determine the intraeellular bactericidal activity of GLS.The data were analyzed by t or t' test.Results The pBudCE4.1/GLS eukaryotic expression recombinant plasmid was successfully constructed.The transcriptional and translational expressions of target gene GLS were detected in RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The bacterial load in macrophages of phorbol myristate acetate(PMA)+pBudCE4.1/GLS group,PMA+pBudCEA.1 group and non-activated group were 1.44±1.25,3.16±0.20 and 3.59±0.21,respectively.The differences between groups were all significant (t=2.403,t=2.854,both P＜0.05).Conclusion Eukaryotic expression recombinant plasmid carrying human GLS gene expressed in macrophages has strong bactericidal activity against intracellular mycobacteria,which provide information for the further study on therapeutic vaccine against Mycobacterium tuberculosis.
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Objective To study the effect of 60 Co radiotherapy on the gene expession of fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) in the cultured rat vascular smooth muscle cells (VSMCs). Methods Rat VSMCs were cultured in DMEM containing 10% FBS, and were radiated by 60 Co at the doses of 0,7,14 and 28Gy, respectively. mRNA level of FN and MMP-2 was measured by RT-PCR. Results The expression level of MMP-2 mRNA decreased from 82.52?6.50 to 64.25?3.81 and 42.17?3.16 in VSMCs with 14,25Gy radiation, respectively(P
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The term "a green hospital", meaning "a safe, dependable and harmless hospital", epitomizes the harmony between the hospital's intrinsic quality and extrinsic surroundings. Thus, the construction of a green hospital possesses rich connotations, viz. powerful guarantee, of medical safety, dependable guarantee of quality, delivery of civilized services and harmonious doctor-patient relationship, and comfortable and humanized surroundings for medical care. It is imperative to formulate comprehensive assessment standards for green hospitals or incorporate such standards into the conditions for hospital appraisal so as to render the idea of green hospital a target of hospital development.
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AIM: To investigate the effects of TGF-?_1 and signal protein Smad3 on rat cardiac myocyte hypertrophy.METHODS: The total protein was analyzed by flow cytometry and the ANF mRNA expression was measured by RT-PCR to judge the hypertrophy of cultured neonatal cardiac myocytes.Smad3 mRNA expression in cardiac myocytes was measured by RT-PCR,and the protein expression of Smad3 was analyzed by Western blotting.RESULTS: TGF-?_1 significantly increased the total protein in cardiac myocytes and promoted ANF mRNA expression,compared with control group.In cultured neonatal myocytes,AS-ODN of Smad3 inhibited myocyte hypertrophy induced by TGF-?_1.Smad3 mRNA and protein expression increased at 15 min after incubated with TGF-?_1,reached the peak at 1 h,and declined at 4 h.CONCLUSION: TGF-?_1 and signal protein Smad3 may participate in the progress of rat cardiac myocyte hypertrophy.