Objective To assess the effects of p27mt gene transfection on the proliferation and apoptosis of human hepatocellular carcinoma cell (HCC) lines SMMC-7721. Methods A replication deficient adenovirus vector encoding p27mt (Ad-p27mt) was used and p27mt cDNA was transfected into human SMMC-7721 cell lines in vitro. The synthesis of DNA in SMMC-7721 cells was determined by using 3H-thymidine incorporation; the cell apoptosis was determined by flow cytometry, TUNEL method and DNA fragmentation analysis. Results The virus titer was 7.95?1012 cfu/ml, the transduction efficiency was 100 % when multiplicity of infection ≥50, FCM analysis revealed a sub-G1 cell peak in Ad-p27mt transduced hepatocellular carcinoma cell lines. Agarose electrophoresis showed marked ladder .The difference of apoptotic index between the Ad-p27mt group and the control group was statistically significant (58.6?4.3, vs 4.5?1.6, P

Objective To observe the effects of Suoquan Wan(SQW) on the endocrine and immune function of the polyuria rats with kidney-yang deficiency. Methods The model rats were induced by gastric infusion of adenine(250 mg/kg) for 4 weeks, then treated respectively with SQW (at low, middle and high dose respectively), Shenqi Wan and desmopressin for another 4 weeks. The bodyweight and organ index were recorded, and serum cortisone concentration were detected. T cell subsets in peripheral blood were determined by flow cytometry. Results The bodyweight of rats in model group was lower than that in the normal control group. And the bodyweight of rats in desmopressin group, middle-and high-dose SQW groups differed from that in the model group (P < 0. 05 or P < 0. 01). The decreased organ indexes of pituitary, adrenal and thymic glands were found in the model group (P < 0. 05 or P < 0. 01 compared with the normal group). The thymus index was elevated in all the medication groups except the low-dose SQW group (P < 0. 05). The elevated organ indexes of pituitary and adrenal glands were only found in Shenqi Wan group and high-dose SQW group (P < 0. 05). In the model group serum cortisone concentration was decreased (P < 0. 01). In Shengqi Wan group and middle-and high-dose SQW groups, the cortisone concentration was significantly increased (P < 0. 05 or P < 0. 01, compared with the model group). In the model group, the percentages of CD3+ and CD3+/CD4+ T cell subsets in pe-ripheral blood were decreased, and the percentage of CD3+/CD8+ T cell subset was increased (P < 0. 05 or P < 0. 01 compared with the normal group). Compared to the model group, the percentages of CD3+ and CD3+/CD4+ T cell sub-sets were increased and the percentage of CD3+/CD8+ T cell subset was decreased in SQW group and Shenqi Wan group. But the differences of T lymphocyte subsets were insignificant between desmopressin group and the model group. Conclu-sion SQW can regulate the endocrine and immune function of the polyuria ratwith kidney-yang deficiency.

Objective:To explore the value of lactic acid (Lac), procalcitonin (PCT), sequential organ failure assessment (SOFA) score and acute physiology and chronic health evaluationⅡ (APACHEⅡ) score in assessing the severity and predicting the prognosis in sepsis shock.Methods:A retrospectively study was conducted. Patients with septic shock hospitalized in the department of critical care medicine of the Affiliated Hospital of Jining Medical University from April 2015 to June 2019 were enrrolled. The patient's gender, age, body mass index (BMI), infection site, organ dysfunction status; Lac, PCT, C-reactive protein (CRP), heart rate and body temperature immediately after admission to the intensive care unit (ICU); APACHEⅡ and SOFA scores within 24 hours, and 28-day prognosis were collected. According to the 28-day prognosis, the patients with septic shock were divided into the survival group and the death group, and the differences in the indicators between the groups were compared. Multivariate Logistic regression analysis was used to screen the risk factors of 28-day death in patients with septic shock; receiver operating characteristic curve (ROC curve) was used to analyze the value of Lac, PCT, SOFA, APACHEⅡ, and age in predicting the 28-day prognosis of patients with septic shock.Results:A total of 303 septic shock patients were enrolled, of which 124 cases survived and 179 died on the 28th day, and the 28-day mortality was 59.08%. ① Compared with the survival group, patients in the death group were older (years old: 66.58±15.22 vs. 61.15±15.68), APACHEⅡ, SOFA, proportion of lung infections, Lac increased [APACHEⅡ score: 22.79±7.62 vs. 17.98±6.88, SOFA score: 9.42±3.51 vs. 5.65±1.59, proportion of lung infections: 53.63% (96/179) vs. 39.52% (49/124), Lac (mmol/L): 5.10±3.72 vs. 3.71±2.56], oxygenation index (PaO 2/FiO 2) and ICU body temperature decreased [PaO 2/FiO 2 (mmHg, 1 mmHg = 0.133 kPa): 198.94±80.15 vs. 220.68±72.06, ICU body temperature (℃): 37.47±1.08 vs. 37.80±1.14], and the differences were statistically significant (all P < 0.05).②Multivariate Logistic regression analysis results: after adjusted for potential confounding factors, APACHEⅡ, PCT, Lac, age and SOFA were independent risk factors for death in patients with septic shock [APACHEⅡ: odds ratio ( OR) =1.05, 95% confidence interval (95% CI) was 1.01-1.10, P = 0.039; PCT: OR = 0.99, 95% CI was 0.98-1.00, P =0.012; Lac: OR = 1.23, 95% CI was 1.08-1.40, P = 0.002; age: OR = 1.03, 95% CI was 1.01-1.05, P =0.009; SOFA score: OR =1.88, 95% CI was 1.59-2.22, P < 0.001]. ③ROC curve analysis showed that APACHEⅡ, Lac, age and SOFA could predict the prognosis of patients with septic shock [APACHEⅡ: the area under the ROC curve (AUC) = 0.682 4, 95% CI was 0.621 7-0.743 1, P = 0.000; when the best cut-off value was 18.500, its sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were 72.63%, 54.84%, 69.89%, 58.12%, 1.608 1 and 0.499 2, respectively. Lac: AUC = 0.604 5, 95% CI was 0.540 8-0.668 2, P = 0.002; when the best cut-off value was 3.550 mmol/L, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were 50.84%, 73.39%, 73.39%, 50.94%, 1.910 3 and 0.669 9, respectively. Age: AUC = 0.599 1, 95% CI was 0.535 4-0.662 7, P = 0.003; when the best cut-off value was 72.500 years old, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were 42.46%, 75.00%, 71.03%, 47.45%, 1.698 3 and 0.767 2, respectively. SOFA: AUC =0.822 3, 95% CI was 0.776 7-0.867 9, P = 0.000; when the best cut-off value was 7.500, its sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were 68.72%, 87.90%, 89.13%, 66.06%, 5.680 4, 0.355 9 respectively]. The combined prediction had a good sensitivity (72.63%) and specificity (84.86%), and AUC (0.876 5) was higher than that of a single variable, suggested that the multivariate combination was more accurate in predicting the short-term outcome of septic shock. Conclusion:Lac, PCT, SOFA score, APACHEⅡ score and age were independent risk factors for death in patients with septic shock, and the accuracy of Lac, SOFA score, APACHEⅡ score and age in predicting short-term prognosis of septic shock was better than that of single variable, and the diagnostic value was higher.

Objective To observe the effects of Suoquan Wan(SQW) on the endocrine and immune function of the polyuria rats with kidney-yang deficiency. Methods The model rats were induced by gastric infusion of adenine(250 mg/kg) for 4 weeks, then treated respectively with SQW (at low, middle and high dose respectively), Shenqi Wan and desmopressin for another 4 weeks. The bodyweight and organ index were recorded, and serum cortisone concentration were detected. T cell subsets in peripheral blood were determined by flow cytometry. Results The bodyweight of rats in model group was lower than that in the normal control group. And the bodyweight of rats in desmopressin group, middle-and high-dose SQW groups differed from that in the model group (P

Background and purpose:In the process of gastric cancer development, cytothesis and apoptosis, and endoplasmic reticulum stress (ERS) are very important pathological processes. Glucose regulated protein 78 (GRP78) and phosphorylated form of extracellular signal-regulated protein kinase (pERK) play important roles in it. This study aimed to investigate the expression of GRP78 and pERK in gastric adenocarcinoma, chronic atrophic gastritis and superficial gastritis, and the role of GRP78 and pERK in the development of gastric adenocarcinoma. Methods:Gastric adenocarcinoma, chronic atrophic gastritis and superifcial gastritis tissues in 60 cases respectively were employed in the study. We chose 25 fresh tissue samples from each group, and the level of GRP78 and pERK mRNA in different tissues were detected by RT-PCR assay. The expressions of GRP78 and pERK in different parafifn samples were detected using immunohistochemistry assay. In addition, the relationships between GRP78 and pERK expression and age, gender, differentiation, invasion, disease stage, and lymphoid node metastasis were analyzed. Results: The expression level of GRP78 and pERK mRNA in gastric adenocarcinoma(1.26±0.18, 2.35±0.36) were significantly higher than chronic atrophic gastritis (0.89±0.25, 1.18±0.25) and superficial gastritis (0.29±0.09, 0.68±0.10, P<0.01). The positive ratio of GRP78 expression in gastric adenocarcinoma, chronic atrophic gastritis and superficial gastritis were 78.3%, 46.6%, 6.7%. The positive ratios of pERK expression were 88.3%, 43.3%, 5.0%, respectively. The GRP78 and pERK expression in different tissues were signiifcantly different (P<0.01). GRP78 and pERK expression were positively correlated with differentiation, disease stage and lymph node metastasis. There was a positive correlation between the gene and protein expression of GRP78 and pERK with a Pearson correlation value of 0.307 and 0.368, respectively. Both univariate and multivariate analysis revealed that GRP78 was related to the prognosis of gastric adenocarcinoma. Conclusion:GRP78 and pERK may play an important role in the transition of normal gastric cells to malignant cells. The expression of these two genes enhances tumor progression. Overexpression of GRP78 and pERK is significantly correlated with poor prognosis in patients with gastric adenocarcinoma. The determination of the expression of GRP78 and pERK might be helpful for the prevention, early diagnosis of gastric carcinoma. Particularly, GRP78 is valuable for the judgement of prognosis, and might be a new target for the treatment of gastric adenocarcinoma.

AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE_3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10.

Objective To establish a rapid and accurate method, and to determine the density of mice and rats sperm with enzyme-labeled instrument.Methods ①The optimal wavelengths and the regression equation set up: After six Kunming mice and six Sprague-Dawley rats were sacrificed, the left epididymis was separated and fully cut up in phosphate buffer saline.With water bath, the sperm were fully dissociated.Using the enzyme-labeled instrument to detect the wavelength absorbance respectively under different wavelength and fitting absorbance curve.The best wavelength will be the most close to 1 of the correlation coefficient ( R2 ) and the standard deviation of minimum.After ten Kunming mice and ten Sprague-Dawley rats were sacrificed,the sperm suspension of different concentration gradient were got.The regression equation of the sperm density and absorbance was established by using enzyme-labeled instrument and haemocytometer.②The test of sperm absorbance stability: Mice and rats,six respectively,were used to make the sperm suspension.Samples were put in room temperature (25℃) or 37℃water bath continued,and after water bath about 0,20,30,40,50, 60 min,the change of absorbance was recorded.③The regression equation verification:The mice were administrated orally with 20% ethanol solution for 30 days to make oligospermia.In order to verify the new method, two different method were used to get the sample sperm.Results The optimal absorbancy-sperm density curve could be established at 380 nm.The means of KM mice sperm count ( x1 ) and absorbance ( y1 ) are showed to be the linear function,and the linear regression equation is y1 =2 ×10 -9 x1 +0.0648, R2 =0.9743.The means of SD rat sperm count ( x2 ) and absorbance (y2) are showed to be the linear function,and the linear regression equation is y2 =5 ×10 -9x2 +0.0621,R2 =0.9940.SD rat sperm suspension liquid after 60 min in water bath, absorbance value at 0 min significantly decreased(P<0.05), but at room temperature after 40 min significantly increased ( P<0.05); KM mice sperm suspension in the water bath and under the condition of normal temperature after 50 min, compared with the 0 min, absorbance value increased significantly(P<0.05).Compared with control group, sperm density of ethanol oligozoospermia group by enzyme standard detector and standard curve calculation were significantly decreased ( P <0.05 );compared with absorbancy-sperm density equation, determination of ethanol oligozoospermia group of sperm density by cell counting plate method had not significant difference.The results suggested absorbancy-sperm density equation could effectively detect the reduction of the mice sperm in oligospermia.Conclusion Using enzyme-labeled instrument to set up the curve of absorbancy-sperm density equation can estimate the sperm density of mice and rats rapidly and exactly.

Objective To evaluate the effect of tirofiban injection in coronary artery occlusion by suction catheter on the opening time of the coronary artery occlusion , the improvement of the blood flow and the incidence of adverse events in 30 days. Methods A total of 97 patients with acute myocardial infarction in recent 4 years were included , whose culprit vessels were subtotal occlusion or total occlusion by angiography and were randomly divided into thrombus aspiration group (group A) and tirofiban injection in occlusion and thrombus aspiration group (group B). The opening time of the coronary artery, the improvement of the blood flow and the incidence of adverse events in 30 days were compared between two groups. Results The opening time of the coronary artery occlusion in group A was shortened when compared with group B but the blood flow arriving TIMI III grade in group B was shorter (P < 0.05). No-reflow incidence in group B was lower while the proportion of blood flow arriving TIMI III grade was higher than that in group A in early phase (P < 0.05). The incidence of adverse events in 30 days was decreased in group B when compared with group A ,but the difference wasn't statistically significant (P > 0.05). Conclusion Direct tirofiban injection in coronary artery occlusion could effectively shorten the opening time of the coronary artery occlusion reduce no-reflow incidence , and improve coronary perfusion but could not decrease the incidence of adverse cardiovascular events in 30 days.

Objective: To investigate the effect of Yi Jin Jing (Sinew-transforming Qigong Exercises) plus tuina on scapulohumeral periarthritis (SP). Methods: A total of 30 cases with SP were randomized into an observation group and a control group. Those in the observation group practiced Yi Jin Jing (Sinew-transforming Qigong Exercises) plus tuina therapy; whereas those in the control group received only tuina therapy. Tuina therapy was conducted every other day, 20 min every time for 1 month and Yi Jin Jing (Sinew-transforming Qigong Exercises) was conducted once a day for 1 month. The therapeutic effects were assessed by visual analogue scale (VAS) and Constant-Murley scale. Results: After treatment, the VAS score and Constant-Murley scale were substantially improved, showing statistical significances (P<0.01); the Constant-Murley scale in the observation group was better than that in the control group, showing a statistical significance (P<0.01); the effective rate in the observation group was higher than that in the control group, between-group comparison showed a statistical significance (P<0.01). Conclusion: Yi Jin Jing (Sinew-transforming Qigong Exercises) plus tuina and tuina alone have a verified effect in treating SP, and the former can achieve a better effect than the later.

Objective To observe the protective effects of Toll-like receptor(TLR)-4 siRNA against acute liver injury in mice induced by lipopolysaccharide(LPS)and D-galactosamine(D-GalN).Methods One hundred and fifty C57BL/6 male mice were divided into 5 groups: phosphate buffered solution(PBS)pretreatment group,negative control plasmid pretreatment group,TS4 pretreatment group,TS6 pretreatment group and TS7 pretreatment group.Acute liver injury was induced in mice by intraperitoneal coinjection of LPS(10 ng/g)and D-GalN(1 mg/g).In vivo delivery of siRNA was performed via the tail vein by hydrodynamic injections(50 μg siRNA dissolved in 1 mL PBS)24 h and 48 h before coinjection of LPS and D-GalN. Expression of TLR-4 in liver tissues was measured by immunohistochemistry.The changes of TLR-4,tumor necrosis factor(TNF)-α and macrophage nflammatory protein(MIP)-2 mRNA levels in liver tissues were determined by reverse transcriptasepolymerase chain reaction(RT-PCR)analysis.MIP-2 and TNF-α concentrations in the sera of mice were determined by enzyme-linked immunosorbent assay(ELISA). Levels of alanine transaminase (ALT) and aspartate transaminase(AST) in serum were measured by standard autoanalyzer techniques. Liver pathological changes were observed by haematoxylin-eosin staining, while cell apoptosis levels in liver were determined by terminal deoxynucleotidyl-mediated-dUTP nick end labeling (TUNEL)assay. The difference of survival rates in 5 groups was analyzed by Fisher's exact probability test.ResultsPretreatment with TLR-4 siRNA down-regulated the TLR-4 mRNA and protein expressions,and significantly decreased the mortality and liver injury caused by coinjection of LPS and D-GalN in C57BL/6 mice.TLR-4 siRNA significantly down-regulated the TNF-α and MIP-2 mRNA expression and cytokine levels as determined by RT-PCR and ELISA,respectively. TLR-4 siRNA abrogated hepatocyte necrosis and inflammatory infiltration and also remarkably reduced serum concentrations of transaminases. The percentage of TUNEL-positive hepatocytes was significantly reduced in TLR-4 siRNA pretreatment group(TS4 pretreatment group: 0.065±0.015 vs PBS pretreatment group; 0.346±0.062,P＜0.05).ConclusionIt suggest that inhibition of TLR-4 expression by TLR-4 siRNA may provide potential application value for preventing liver injury.