BACKGROUND: Compared to those original viruses systems, adeasy adenovirus, a recombinant adenoviral system widely used in recent years, based on viruses with a deletion of both El and E3, reported by T.C. He in 1998, is an improved one. It simplifies the generation and production of such viruses and expedite the process of generating and testing recombinant adenoviruses using homologous recombination in bacteria rather than in eukaryotic cells. Moreover, it can be conveniently followed with the aid of green fluorescent protein encoded by EGFP gene incorporated into the viral backbone.OBJECTIVE: To construct the recombinant adenovirus and to evaluate them by transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.

Objective To investigate the curative effect of bronchoalveolar lavage with fiberoptic bronchoscopy combined with vibration sputum drainage in the treatment of severe pneumonia patients undergoing mechanical ventilation (MV).Methods A prospective randomized controlled trial was conducted. 286 severe pneumonia patients undergoing MV admitted to intensive care unit (ICU) of Hunan People's Hospital from January 2014 to July 2016 were enrolled, and they were divided into control group and observation group according to random number table, with 143 patients in each group. Patients in both groups received sensitive antibiotics for anti-infection, etiological treatment, and calefacient and humidifying treatment. The patients in the control group received bronchoalveolar lavage with fiberoptic bronchoscopy, and those in the observation group received bronchoalveolar lavage combined with vibration sputum drainage. The parameters of respiratory function and inflammation before and after treatment, curative effect, and prognosis were compared between the two groups.Results ① There were no significant differences in respiratory function parameters between the two groups before treatment, 2 hours after treatment, the parameters were improved in both groups. Moreover, oxygenation index (PaO2/FiO2) in observation group was significantly higher than that of control group [mmHg (1 mmHg = 0.133 kPa): 379.1±20.2 vs. 351.8±24.7], and arterial partial pressure of carbon dioxide (PaCO2) and airway resistance (Raw) were significantly lower than those of the control group[PaCO2 (mmHg): 36.5±5.8 vs. 45.3±6.9, Raw (cmH2O, 1 cmH2O = 0.098 kPa): 12.9±0.6 vs. 13.1±0.8, allP < 0.01]. ② There were no significant differences in inflammation parameters between the two groups before treatment, 24 hours after intervention, which were significantly decreased in both groups. Moreover, white blood cell count (WBC), procalcitonin (PCT) and C-reactive protein (CRP) in the observation group were significantly lower than those of the control group [WBC (×109/L): 8.2±1.7 vs. 12.8±3.7, PCT (μg/L): 15.4±2.4 vs. 21.8±3.1, CRP (mg/L): 37.1±6.1 vs. 67.2±7.2, allP < 0.01]. ③ Compared with the control group, the treatment efficiency of observation group was improved [95.1% (136/143) vs. 87.4% (125/143)], the quantity of sputum excretion was increased (mL: 49.2±12.5 vs. 36.9±11.0), duration of MV and length of ICU stay were significantly shortened (days: 6.4±3.6 vs. 9.4±2.1, 8.6±5.7 vs. 12.4±4.6, bothP < 0.01), however, there was no significantly statistical difference in 28-day mortality between control group and observation group [2.8% (4/143) vs. 2.1% (3/143),P > 0.05].Conclusion Compared with bronchoalveolar lavage with fiberoptic bronchoscopy alone, the treatment of bronchoalveolar lavage combined with vibration sputum drainage is more effective in sputum excretion for severe pneumonia patients undergoing MV, which could improve the respiratory function, reduce infection, shorten the duration of MV and the length of ICU stay, and improve the recovery.

Objective: To observe the effect of moxibustion on behaviors and related products of tryptophan (Trp) metabolism in the colon of mice with irritable bowel syndrome (IBS), and to explore the mechanism of moxibustion in the IBS treatment.Methods: Twenty-four mice were randomly divided into a normal group, a model group, a moxibustion group, and a probiotic group, with 6 mice in each group. The visceral pain model of IBS was established by enema with 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution. Mice in the moxibustion group were treated with mild moxibustion at bilateral Zusanli (ST36), and those in the probiotic group were treated with probiotics such as Bifidobacterium by gavage. Abdominal withdrawal reflex (AWR) test, elevated plus-maze (EPM) test, and forced swimming test (FST) were performed after treatment. The expression levels of 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (TPH1) in the colon were detected by immunofluorescence, and the expression levels of Trp, kynurenine (Kyn), and indole-2,3-oxygenase (IDO) in the colon were detected by enzyme-linked immunosorbent assay. Results: Compared with the normal group, the AWR scores were increased significantly in the model group under different pressure values (P<0.01), the open-arm staying time and open-arm entries in the EPM test were decreased significantly (P<0.01, P<0.05), the motionless time in the FST was increased significantly (P<0.01), and the expression levels of colonic Trp, TPH1, IDO, 5-HT, and Kyn were increased significantly (P<0.01) in the models. Compared with the model group, the AWR scores were differently decreased (P<0.05 or P<0.01), the open-arm entries in the EPM test were increased (P<0.05), the motionless times in the FST were decreased (P<0.05), and the colonic expression levels of Trp, TPH1, IDO, and 5-HT were decreased (P<0.01 or P<0.05) in the moxibustion and probiotic groups; the open-arm staying time was significantly increased in the moxibustion group (P<0.01), and the colonic expression level of Kyn was significantly decreased in the probiotic group (P<0.01). Conclusion: Moxibustion at Zusanli (ST36) improves visceral pain and pain mood and down-regulates the expression levels of colonic TPH1, IDO, Trp, 5-HT, and Kyn in IBS mice.

Objective To characterize the viability and transgene expression of articular chon-drocytes cultured in 3-Dimensional scaffolds provided by four types of carriers.Methods Articular chondrocytes from rabbit knees were cultured and infected with adenovirus that could express green fluo-rescence protein (AdGFP) and GL3 luciferase (AdGL3-Luc).The viability and gene expression were determined with fluorescence microscopy and luciferase assays in four types of scaffolds;type I collagen sponge, fibrin glue, hyaluronan and open-cell polylactic acid (OPLA).Cartilage matrix production was assessed by Alcian blue staining.Results Articular chondrocytes of rabbits were effectively infected by AdGFP and exhibited sustained GFP expression.All the tested scaffolds supported the survival and gene expression of the infected chondrocytes.However, the highest transgene expression was observed in the OPLA carrier (P<0.01).Alcian blue-positive matrix materials were readily detected in OPLA cultures four weeks later.Conclusion OPLA supports the highest transgene expression and is the most conduc-tive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repair.

Objective To explore the superiority of dual-phase contrast-enhancement multi-slice computed tomography (MSCT) in observing intracranial tuberculosis.Methods Thirty patients with intracranial tuberculosis,admitted to our hospital from January 2003 to November 2011,were chosen in our study; regular 16-slice spiral CT scan was performed,and then,dual-phase MSCT was performed as follows:contrast-enhanced scan was performed 25 seconds after arrival of contrast material on vascular phase,and 5 minutes after that (lag phase),conventional scanning was performed.According to the different lesions and morphologies of the tuberculosis,they were divided into three types:meningeal thickening,meningeal tuberculoma and parenchymal tuberculoma; the lesion sizes and edge definition and the relationship of the lesions with the surrounding vascular lesions in these three types were scored and calculated,and the differences of the image quality were compared at the vascular phase and lag phase.Results A total of 526 lesions in 30 patients were found,including 22 with meningeal thickening,235 with meningeal tuberculomas/tubercles,and 269 with parenchymal tuberculomas/tubercles.As compared with these three types at the vascular phase (0.36±0.49,0.36±0.52 and 0.41±0.53; 0.00,0.27±0.45 and 0.12±0.32; 1.09±0.68,1.22±0.74 and 1.27±0.75),these three types at the lag phase had significant differences in the scores of lesion sizes (1.64±0.58,1.64±0.58 and 1.59± 0.60) and lesion edge definition (2.00,1.73±0.49 and 1.88±0.34) and the relationship of the lesions with the surrounding vascular lesions (1.82±0.39,2.00±0.06 and 2.00±0.06,P＜0.05).Conclusion Images in the lag phase have advantages on diagnosis of intracranial tuberculosis.

Objective: To explore the effects of lncRNA HULC on hepatocellular carcinoma (HCC) growth by down-regulating miR-29. Methods: The expression levels of HULC and miR-29 in HCC tissues and cells were detected by real-time quantitative PCR (RT-qPCR), and the correlation analysis was performed. After HCC cells were transfected with HULC overexpressed plasmid or siRNA, the expressions of miR-29 and its target gene SETDB1 were determinate by RT-qPCR. According to the bioinformatic prediction of the miR-29 binding site in the HULC sequence, the report gene plasmids were constructed. The HCC cells were co-transfected with miR-29 mimics or miR-29 inhibitor, and the HULC targeted regulation of miR-29 was verified by dual luciferase reporter assay. The effect of miR-29 on the HULC-mediated proliferation in HCC cells was detected by cell count kit 8 (CCK-8) experiment. Expression of tumor proliferation antigen Ki-67 was detected by RT-qPCR.The Hep3B cells were inoculated in mice and miR-29 mimics and miR-29 negative control (NC) further injected into the lesions. The tumor volume was observed, and the expressions of tumor proliferation antigen ki-67 in tumor tissues were detected by immunohistochemical staining. Results: The expression of HULC was significantly up-regulated while the expression of miR-29 was significantly down-regulated in HCC tissues and cells (P<0.01). The level of HULC was negatively correlated with miR-29 in tumor tissues (r=-0.754, P<0.01) and HCC cells (r=-0.865, P<0.05). The in vitro experiments showed that, compared with the blank control group, the expression of miR-29 in HULC overexpressed Huh7 cells was significantly reduced, while the mRNA level of miR-29 target gene SETDB1 was increased (P<0.01). The expression of miR-29 was significantly increased in HULC deleted Hep3B cells, while the mRNA expression of SETDB1 was decreased (P<0.01). Double luciferase reporter gene assay showed that miR-29 mimics significantly inhibited the luciferase activity of Hep3B cells transfected with HULC wide type (psi-HULC-WT) plasmid but had no effect on Hep3B cells transfected with mutant plasmid (psi-HULC-Mut). However, the miR-29 inhibitor antagonized the inhibitory effect of miR-29 mimics on luciferase activity of psi-HULC-WT (P<0.01). Cell proliferation experiments showed that, compared with the control group, the proliferation ability of miR-29 mimics overexpressed Huh7 cells was significantly reduced.After 24, 48 and 72 hours of treatment, the proliferation rates of Huh7 cells in the HULC overexpressed group were (43.87±3.82)%, (83.45±7.46)% and (123.34±8.67)%, respectively, significantly higher than (13.45±1.77)%, (23.54±1.37)% and (38.21±2.09)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of miR-29 mimics transfected Huh7 cells were (57.10±1.94)% and (73.76±3.46)%, respectively, significantly lower than (83.45±7.46)% and (123.34±8.67)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of Huh7 cells transfected with miR-29 mimics and miR-29 inhibitor group were (76.45±3.24)% and (89.37±4.37)%, respectively, significant higher than (57.10%±1.94)% and (73.76±3.46)% of the control group (P<0.05). After 48 h transfection, the expression of Ki-67 in Huh7 transfected with miR-29 mimics was significantly inhibited compared with the control group (P<0.01). However, the expression of Ki-67 mRNA was increased in Huh7 cells transfected with miR-29 inhibitor (P<0.01). The results of in vivo experiments showed that the tumor volumes of the control group, miR-29 mimics group and miR-29 mimics + miR-29 inhibitors group were (504.0±19.6) mm³, (310.0±24.3) mm³ and (483.7±21.2) mm³, respectively. Injection of miR-29 mimics reduced while miR-29 inhibitor promoted tumorigenesis ability of Huh7 in nude mice (P<0.01). The immunohistochemical staining showed that the average optical density values of Ki-67 protein in tumor tissues of the control group, miR-29 mimics group and miR-29 analogue+ miR-29 inhibitor group were 0.65±0.08, 0.36±0.07 and 0.56±0.06, respectively. The expression level of Ki-67 protein in miR-29 mimics group was significantly reduced (P<0.01) while increased in the miR-29 mimics+ miR-29 inhibitor group (P<0.01). Conclusion LncRNA HULC promotes HCC growth by down-regulating miR-29.

Objective: To investigate the application value of real-time virtual sonography(RVS)in the diagnosis and treatment of complicated hepatolithiasis. Methods: The retrospective and descriptive study was conducted. The clinical data of 10 patients with complicated hepatolithiasis who were admitted to Hunan Provincial People′s Hospital between October 2017 and March 2018 were collected. There were 3 males and 7 females, aged from 40 to 69 years, with an average age of 57 years. Patients received abdominal color Doppler ultrasound examination, magnetic resonance cholangiopancreatography, and upper abdominal spiral computed tomography (CT) thinly scanning + enhanced examination. Data of CT examination were imported into RVS. RVS was used to locate hepatolithiasis, relationship between stones and vessels, anatomy of bile ducts and vessels in hepatic hilus. Surgical methods included RVS-guided hilar cholangiotomy, biliary stricturoplasty, bilateral hepatojejunostomy, hepatic segmentectomy (lobectomy), and hepatolithotomy. Observation indicators: (1) surgical and postoperative situations; (2) typical case analysis; (3) follow-up. Follow-up using outpatient examination was performed to detect residual stones up to June 2019. Measurement data with skewed distribution were represented as M(range). Count data were described as absolute numbers. Results: (1) Surgical and postoperative situations: 10 patients underwent RVS-guided surgeries successfully for complicated hepatolithiasis, with successful match in RVS (difference between CT images and ultrosound images <2 mm). No residual stone was identified by choledochoscope during operation. The operation time and volume of intraoperative blood loss were 285 minutes (range, 210-360 minutes) and 200 mL (range, 100-600 mL), respectively. No blood transfusion was needed during the operations. The duration of hospital stay was 20.5 days (range, 14.0-29.0 days). There was no perioperative death. One patient had postoperative biliary leakage and abdominal infection, and was cured after conservative treatment. (2) Typical case analysis: the tenth patient, female, 60 years old, was diagnosed with complicated hepatolithiasis, and was prepared to undergo hepatolithotomy+ quadrate lobectomy and hilar cholangioplasty+ bilateral hepatojejunostomy. Preoperative CT images and intraoperative color Doppler ultrasound images of the patient were fused and matched on the sagittal section of the portal vein and the cross section of the right branch of portal vein, and stones and important vessels were marked on the images. After accurate positioning, a curette was used to remove the stones. Removal of biliary stones through hepatic parenchyma and peripheral dilated bile ducts was conducted at the site where stones obviously existed. After the stones were removed, the intrahepatic bile duct and hilar bile duct merged. The left end of the bile duct split was confirmed by real-time ultrasound. After location of portal vein was determined by ultrasound, vascular plastic surgery was perfomed to avoid stenosis. (3) Follow-up: 10 patients were followed up for 6-12 months, with a median follow-up time of 8 months. One of 10 patients was suspected residual stones at the right peripheral hepatic anterior lobe by postoperative angiography at 2 months after surgery, and was not removed stones by choledochoscope. The patient had no recurrent symptoms after T-tube removal. The other 9 patients had no residual stones. Conclusion RVS applied in complicated hepatolithiasis is helpful for the precise intraoperative diagnosis, and the surgical treatment can be safe and effective.