Objective:To construct recombinant expressing vector pcDNA3-DAF and to develop the NIH3T3 cell model expess human complement regulatory protein decay accelerating factor(DAF,CD55)stably after transfected.Methods:Human membrane complement regulatory protein(hCRP) DAF cDNA containing the full-length of encoding region was cloned into expressing vector pcDNA3.After identification by restriction enzyme digestion,PCR and sequencing,the recombinant plasmid was transfected into NIH3T3 cells with calcium phosphate-DNA precipitate method.A stably-transfected cell line was established by G418 selection.Extraneous gene integration was identified by PCR.Expression of DAF at both mRNA and protein levels was analyzed by RT-PCR,Western blot and indirect immunofluorescence microscopy.Results:The eukaryotic expression vector pcDNA3-DAF was successfully constructed and the DAF gene was transfected stably into NIH3T3 cells,a stably-transfected cell line was established and DAF was efficiently expressed on the surface of transfected NIH3T3 cells.Human DAF cDNA was integrated into NIH3T3 pcDNA3-DAF genomic DNA after continuous 30 times passages,indicating that NIH3T3 pcDNA3-DAF was stable cell line.Conclusion:The establishment of the stably-transfected cell line and the expression of the target gene provide a base for further studies on the function of the DAF and the cooperative fashion among different human complement regulatory proteins in alleviating the complement-mediated cytolysis.

Objective To investigate the accuracy of the plasma DNA concentration in evaluating the prognosis in patients with sepsis.Methods One hundred and sixty patients with sepsis were enrolled as the sepsis group (group SE).Another 109 patients without sepsis hospitalized during the same period served as the control group (group C).The venous blood sample was taken on admission for determination of plasma DNA concentration by polymerase chain reaction,C reactive protein (CRP) concentration by ELISA.APACHE Ⅱ score and SOFA score were evaluated at 24 h after admission.The 160 patients with sepsis were divided into two groups according to the result of prognosis:survival group ( n =103) and death group ( n =57).Results Compared with group C,the plasma DNA concentration,CRP concentration,APACHE Ⅱ score and SOFA score were significantly increased in group SE (P＜0.05).Compared with survival group,the plasma DNA concentration,APACHEⅡ score and SOFA score were significantly increased in death group ( P ＜ 0.05).The areas under receiver operating characteristic (ROC) curves of the plasma DNA concentration was significantly larger than those of APACHE Ⅱ score and SOFA score (0.81(95％ CI,0.74-0.88) versus 0.68(95％ CI,0.60-0.77),or 0.72(95％ CI,0.63-0.82)).Conclusion The plasma DNA concentration can accurately evaluate the prognosis in patients with sepsis.As compared with the plasma CRP concentration,APACHE Ⅱ score and SOFA score,the plasma DNA concentration is more accurate to evaluate the prognosis in patients with sepsis.

Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP were protected from C-mediated damage and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack, which was compared with either DAF or MCP alone. These results suggest that the dicistronic vector could improve the efficiency of multi-gene delivery and benefit the synergic effect of human membrane complement regulatory proteins DAF and MCP.
3T3 Cells ; Animals ; CD55 Antigens ; biosynthesis ; genetics ; pharmacology ; DNA, Complementary ; genetics ; Drug Synergism ; Graft Rejection ; prevention & control ; Humans ; Membrane Cofactor Protein ; biosynthesis ; genetics ; pharmacology ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection

Objective:To compare the clinical efficacies of Da Vinci robotic and laparoscopic total mesorectal excision (TME) for low rectal cancer.Methods:The retrospective cohort study was conducted. The clinicopathological data of 64 patients with low rectal cancer who were admitted to the Third Xiangya Hospital of Central South University from October 2015 to January 2019 were collected. There were 42 males and 22 females, aged from 40 to 84 years, with a median age of 59 years. Of the 64 patients, 31 undergoing Da Vinci robotic TME and 33 undergoing laparoscopic TME were allocated into robotic group and laparoscopic group, respectively. Observation indicators: (1) surgical situations and postoperative recovery; (2) postoperative pathological examination; (3) follow-up. Follow-up using outpatient examination and telephone interview was conducted to detect long-term complications and pelvic autonomic nerve injury up to January 2020. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was analyzed using the t test. Measurement data with skewed distribution were represented as M ( P25, P75), and comparison between groups was analyzed using the rank sum test. Count data were represented as absolute numbers, and comparison between groups was analyzed using the chi-square test or Fisher exact probability. Comparison of ordinal data between groups was analyzed using the Mann-Whitney U test. Results:(1) Surgical situations and postoperative recovery: cases with anus preservation, cases with defunctioning stoma, cases with intraoperative conversion to laparotomy, cases with intraoperative blood transfusion, operation time, volume of intraoperative blood loss, time to postoperative first out-of-bed activities, time to postoperative first flatus, time to postoperative diet resumption, duration of hospital stay, cases with incision infection, cases with postoperative hemorrhage, cases with anastomotic fistula, cases with pulmonary infection, cases with urinary retention, comprehensive complication index for the whole group, comprehensive complication index for patients with complications, and treatment expenses of the robotic group were 30, 23, 0, 1, (285±73)minutes, (147±112)mL, (1.6±0.8)days, (3.6±1.1)days, (3.2±1.5)days, (9.8±2.8)days, 1, 4, 3, 1, 4, 15.0±12.0, 22.6(20.9, 27.3), (11.7±1.2)×10 4 yuan, respectively. The above indicators of the laparoscopic group were 22, 13, 2, 2, (253±57)minutes, (211±123) mL, (1.8±0.8) days, (3.9±1.6)days, (4.1±1.9)days, (11.8±4.3)days, 2, 5, 3, 3, 2, 20.0±12.0, 24.2(10.5, 30.8), (7.7±1.3)×10 4 yuan, respectively. There were significant differences in the cases with anus preservation, volume of intraoperative blood loss, duration of hospital stay, and treatment expenses between the two groups ( χ2=8.581, t=-2.065, -2.133, 12.700, P<0.05). There was no significant difference in the cases with defunctioning stoma, operation time, time to postoperative first out-of-bed activities, time to postoperative first flatus, time to postoperative diet resumption, comprehensive complication index for the whole group, or comprehensive complication index for patients with complications between the two groups ( χ2=2.425, t=1.957, -0.679, -0.846, -1.941, -1.867, Z=-0.850, P>0.05). There was no significant difference in the cases with intraoperative conversion to laparotomy, cases with intraoperative blood transfusion, cases with incision infection, cases with postoperative hemorrhage, cases with anastomotic fistula, cases with pulmonary infection, or cases with urinary retention between the two groups( P>0.05). One patient with anastomotic fistula in the robotic group was clipped under endoscopy, and the other patients with complications were cured after symptomatic treatment. (2) Postoperative pathological examination: distance from tumor to surgical margin, tumor diameter, case with positive or negative surgical margin, cases with highly, highly-moderately, moderately, moderately-poorly, poorly differentiated tumor (tumor differentiation degree), cases in stage Ⅰ, Ⅱ, Ⅲa+ b, Ⅲc+ Ⅳ (tumor pathological stage), the number of lymph node dissected were 1.0 cm(0.3 cm, 2.0 cm), (3.5±1.1)cm, 2, 29, 3, 7, 14, 5, 2, 5, 18, 4, 4, 16.0±2.8 of the robotic group, respectively, versus 1.3 cm(0.5 cm, 3.0 cm), (4.2±1.4)cm, 2, 30, 1, 7, 16, 6, 3, 1, 19, 7, 6, 13.9±3.8 of the laparoscopic group. There was a significant difference in the number of lymph node dissected between the two groups ( t=2.420, P<0.05) . There was no significant difference in the distance from tumor to surgical margin, tumor diameter, tumor differentiation degree, or tumor pathological stage between the two groups ( Z=-0.980, t=-1.912, Z=-0.809, -1.595, P>0.05). There was no significant difference in the surgical margin between the two groups ( P>0.05). (3) Follow-up: of the 31 patients in the robotic group, 29 were followed up for 3-24 months, with a median follow-up time of 12 months. Of the 33 patients in the laparoscopic group, 30 were followed up for 3-36 months, with a median follow-up time of 15 months. Cases with intestinal obstruction, cases with timely stoma closure, cases with local recurrence, cases with distant metastasis, cases with death, Wexner score at postoperative 12 months, international prostate symptom score at postoperative 12 months, times of nocturia at postoperative 12 months, international index of erectile function of the robotic group were 2, 20, 3, 2, 2, 0.0(0.0, 0.0), 4.5(1.3, 8.8), 1.5(1.0, 2.0), 2.0(1.3, 10.8), respectively. The above indicators were 4, 7, 3, 2, 3, 1.0(0.0, 3.0), 8.0(2.0, 14.3), 2.0(1.0, 4.0), 3.0(1.0, 11.8) of the laparoscopic group. There was no significant difference in the cases with intestinal obstruction, cases with timely stoma closure, cases with local recurrence, cases with distant metastasis, or cases with death between the two groups ( P>0.05). There were significant difference in the Wexner score and times of nocturia at postoperative 12 months between the two groups ( Z=-2.202, -1.986, P<0.05). There was no significant difference in the international prostate symptom score and international index of erectile function at postoperative 12 months between the two groups ( Z=-0.885, 0.094, P>0.05). Conclusion:Both Da Vinci robotic and laparoscopic TME for low rectal cancer are safe and effective, of which the former can improve the anal sphincter retention rate, reduce the nocturia frequency and enhance the protection of defecation function under the premise of radical resection of tumor.

Objective To investigate the correlation between synovial inflammatory changes (synovial blood signals,synovial thickness,joint effusion) and cartilage injury,meniscus prominence of knee osteoarthritis (KOA) under energy Doppler ultrasound and further to assess the value of ultrasound for prognostic judgment in KOA.Methods A total of 291 KOA patients from our hospital from 2016 to 2018 were collected.The patients with rheumatoid arthritis (RA) with knee joints involved were recruited as control group.The data that conform to the normal distribution were expressed by mean±SD,and others were represented by M (P25,P75).The t test,the rank test and Chi-square test were used between the two groups,and ANOVA or K-W test was used to compare the data between the multiple groups,Spearman correlation analysis was used for correlation analysis.Results ① The proportion of synovial blood flow signal in KOA group was lower than RA group (19.6％ vs 47.7％,x2=286.2,P＜0.01),as well as the typical synovial thickness [2.6(0,3.95) mm vs 3.43 (1.85,6) mm,Z=-3.674,P＜0.01] and popliteal cyst (11.5％ vs 18.5％,x2=4.484,P=0.04).② In KOA group,no significant difference was found between ESR and hs-CRP among different synovial blood signals (H=7.213,H=0.883,all P＞0.05).ESR from KOA group were both significantly lower than controls but the synovial blood signals was the same except for power Doppler signal-2 group (Z=-8.414,Z=-4.991,t=-3.428,all P＜0.05);hs-CRP from KOA group were both significantly lower than controls but the synovial blood signals was the same.③ A total of 489 knee joints were detected in 291 patients with KOA,of which synovial blood flow signals were found in 96 joints,the power Doppler signal-I group was the most frequent in KOA (80/96,83.3％).In addition,joint effusion and synovial thickening were positively correlated with the synovial blood signals,(r=0.277,r=0.411,all P＜0.05).④ At last,the degrees of ultrasound blood flow signal in KOA group was positively associated with the WOMAC score,the Lequsne index and US-1 score (r=0.352,r=0.424,r=0.59,all P＜0.05).Conclusion Energy Doppler ultrasonography can be used to detect the KOA synovitis.Synovitis is not rare in KOA patients,and which is associated with disease activity.However,KOA and RA cannot be differentiated by energy Doppler ultrasonography.

Objective To analyze the risk factors of prognosis in elderly patients with septic shock in ICU.Methods Clinical data of 113 elderly patients with septic shock admitted in the ICU of Ningbo Medical Center Lihuili Eastern Hospital from November 2015 to March 2019 were retrospectively analyzed. Among them, 40 patients died ( death group) and 73 patients survived (survival group) within 28 d after diagnosis confirmed.The general information ,underlying disease,laboratory findings and invasive operations were compared between the two groups.Multivariate Logistic regression was used to analyze the risk factors for prognosis of patients, and ROC curve was used to analyze the predictive value of risk factors for death . Results Multivariate Logistic regression analysis showed that APACHEII score ( OR ＝1.344, 95%CI 1畅187-1.520,P＜0.01) and lactic acid level at admission (OR＝1.311,95%CI 1.075-1.599,P＜0.01) were independent risk factors for prognosis ,while platelet counts (OR＝0.986,95%CI 0.976-0.996,P＜0畅01)and albumin level(OR＝0.812,95%CI 0.697-0.945,P＜0.01)were protective factors for prognosis. ROC curve analysis showed that the area under the curve of APACHEII score ,lactic acid level at admission and APACHEII score combined with lactic acid level at admission were 0.861(95%CI 0.784-0畅919,P＜0畅01),0.752(95%CI 0.662-0.828,P＜0.01) and 0.904(95%CI 0.834-0.951,P＜0.01),respectively. The predictive value of APACHEII score combined with lactic acid level at admission was better than those of APACHEII score and lactic acid level at admission ( Z ＝2.175 and 2.879, P ＜0.05 and P ＜0畅01). Conclusions Lower APACHEII score and lactic acid level ,increased platelet counts and albumin level may be associated with a favorable prognosis for elderly patients with septic shock .APACHEII score combined with lactic acid level at admission has better predictive value for prognosis .

Objective To analyze the risk factors of renal replacement therapy (RRT) in acute kidney injury (AKI) patients after liver transplantation, and to investigate the prognosis effect of initial RRT treatment time. Methods Clinical data of 132 recipients undergoing organ donation for cardiac death (DCD) allograft orthotopic liver transplantation admitted to Ningbo Medical Center Lihuili Hospital and Ningbo Medical Center Lihuili Eastern Hospital from July 2014 to July 2018 was retrospectively analyzed. AKI was defined and staged by the criteria of Kidney Disease Improving Global Outcomes (KDIGO) guideline in the first 7 days. According to the implementation of RRT, the patients were divided into non-RRT group and RRT group. The differences in gender, age, body mass index (BMI), model for end-stage liver disease with serum sodium (MELD-Na) score, serum creatinine (SCr), and intraoperative norepinephrine (NE) dose, blood loss, fluid infusion, anhepatic phase time, duration of operation between two groups were compared. The statistically significant risk factors of AKI found by univariate analysis were selected and analyzed to find independent risk factors of RRT in AKI patients after liver transplantation with multivariate Logistic regression analysis. The receiver operating characteristic (ROC) curve was drawn to evaluate the test efficiency of all risk factors of RRT implementation. According to the implementation of RRT on KDIGO stage-2, all the patients on KDIGO stage-2 and stage-3 were divided into early group (initial RRT on KDIGO stage-2) and delayed group (including self-improvement without RRT on KDIGO stage-2 and initial RRT on KDIGO stage-3). The duration of mechanical ventilation, the length of intensive care unit (ICU) stay, AKI duration, incidence of catheter related bloodstream infection (CRBSI) and 28-day mortality were compared between the two groups. Results All 132 receptors were enrolled in the final analysis, and 77 patients developed AKI, accounting for 58.3%, among which 52 cases were in RRT group (67.5%) and 25 were in non-RRT group (32.5%). As shown by univariate analysis, the MELD-Na score (21.6±4.4 vs. 18.0±4.3), intraoperative NE dose (μg·kg-1·h-1: 7.5±1.2 vs. 5.2±1.7), blood loss [mL: 3 000 (2 200, 4 000) vs. 2 600 (1 800, 3 200)], fluid infusion [mL: 6 400 (4 500, 7 800) vs. 5 600 (4 200, 6 800)], and anhepatic period (minutes: 65.6±4.5 vs. 63.0±5.0) were significantly increased in RRT group as compared with those in non-RRT group (all P < 0.05). There was no significant difference in gender, age, BMI, SCr before operation or the duration of operation. It was shown by multivariate Logistic regression analysis that MELD-Na score before operation [odds ratio (OR) = 1.398, 95% confidence interval (95%CI) = 1.062-1.841, P = 0.017], intraoperative NE dose (OR = 4.724, 95%CI = 2.036-10.961, P = 0.000) and fluid infusion (OR = 1.002, 95%CI = 1.001-1.004, P = 0.010) were independent risk factors of RRT implementation in AKI patients after liver transplantation. It was shown by ROC curve analysis that the area under the ROC curve (AUC) of MELD-Na score, NE dose and fluid infusion for predicting the implementation of RRT in AKI patients after liver transplantation was 0.719, 0.867, and 0.670, respectively, which suggesting that NE dose had moderate predictive value, but MELD-Na score and fluid infusion had low predicative value. When the optimal cut-off value of NE dose was 6.5 μg·kg-1·h-1, the sensitivity was 84.6% and the specificity was 80.0%. The 28-day mortality was both 0 in early group (n = 25) and delayed group (n = 39). Compared with the early group, the duration of mechanical ventilation (hours: 41.0±1.0 vs. 35.8±6.7) and the length of ICU stay (hours: 98.8±6.6 vs. 94.2±7.3) were significantly increased in delayed group (both P < 0.05), there was no significant difference in AKI duration (days: 11.8±4.2 vs. 10.6±4.9) or the incidence of CRBSI [5.1% (2/39) vs. 4.0% (1/25), both P > 0.05]. Conclusions MELD-Na score, intraoperative NE dose and fluid infusion were the independent risk factors of RRT implementation in AKI patients after liver transplantation. NE dose had moderate predictive value, but MELD-Na score and fluid infusion had low predicative value. Initial RRT on KDIGO stage-2 could reduce the duration of mechanical ventilation and the length of ICU stay.