OBJECTIVETo establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein.

METHODSWe deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm.

RESULTSThe optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962.

CONCLUSIONThe BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.

Animals ; Antibodies ; Cystic Fibrosis Transmembrane Conductance Regulator ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Humans ; Peptides ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity

Through an overview of the development of long-term care mode that combined medicine and pen-sion and the existing ethical issues, this paper pointed out that the future long-term care mode should strengthen the enthusiasm of public hospitals to participate in, reduce multiple management by government departments, es-tablish perfect laws and regulations and medical insurance system, enhance the responsibility of the practitioners and the general public, meanwhile, actively practice the principle of humanity and respect, fairness and efficiency, honesty and reliability in ethics, to provide new ideas and directions for the development of this model.

AIM To investigate the effect of recipient kidney function by CsA coadministration Ber used to induce immune tolerance in rats of allogenic cardiac transplantation. METHODS The authors established the SD to Wistar rats heterotopic cardiac transplantation model by Onos methods.Observe the cardiac allograft survival and levels of BUN and Cr in the recipients plasma. The recipients were classified into 5 groups randomly after heterotopic cardiac transplantation were performed. Group A (Wistar to Wistar)): Received placebo intraperitoneal injected for 21 days; Group B (SD to Wistar): Saline intraperitoneal injected for 21 days; Group C (SD to Wistar):CsA 2 mg?kg -1 ?d -1 intraperitoneal injected for 21 days; Group D(SD to Wistar):Ber 16 mg?kg -1 ?d -1 gastrointubation for 21 days; Group E(SD to Wistar): Ber 16 mg?kg -1 ?d -1 gastrointubation coadministration CsA 2 mg?kg -1 ?d -1 ip for 21 days. RESULTS The levels of BUN and Cr in recipint plasma is lower evidently compare with the group with CsA ip simply. CONCLUSION Ber can reduce the renal toxicity in recipients by CsA which was intraperitoneal injected (ip) over a long period time.

In this study, we investigated the pharmacokinetics parameters of SPIO-shRNA dual functional molecular probe and observed the main organ distribution by MRI in vivo. Eighteen New Zealand white rabbits were randomly divided into three groups and injected intravenously with different doses of SPIO-shRNA molecular probe, respectively. The blood samples were collected to analyze the pharmacokinetic parameters by measuring the iron content at 30 minutes before and after the injection. Twenty-four Kun Ming (KM) mice were randomly divided into 4 groups: the control group was injected intravenously with physiological saline 200 µL per mouse via the tail vein, the other 3 groups were injected intravenously with different doses of SPIO-shRNA molecular probe. MRI observation was performed in 24 hours, and the liver, spleen, kidney, brain and muscle were collected for iron quantification with Prussian blue staining to determine distribution of the SPIO-shRNA molecular probe in the main organ in vivo. Our results suggest that the molecular probe blood half-life is more than 3 hours. The data of MRI suggest the probe was distributed in liver and spleen, and the MRI signal was reduced with the increase in probe's doses (P < 0.05). The results of Prussian blue staining confirmed the results of MRI. Most of the probe could escape the phagocytosis of mononuclear phagocyte system. Our data provide the pharmacokinetic and distribution of SPIO-shRNA molecular probe in organs. Meanwhile, it suggests the choice of the time and dose of probe for MR imaging of tumor in vivo.
Animals ; Half-Life ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; Mice ; Molecular Probes ; pharmacokinetics ; RNA, Small Interfering ; chemistry ; Rabbits

Objective To know the quality of iodized salt and the current situation of the salt coverage in Tibet,and to provide scientific basis for proposing proper prevention and control measures to Iodine dificiency disorders(IDD). Methods In 2008, according to the "Sampling Methods of the Main Products in the Salt Industry",one batch fifteen salt samples were collected in iodized salt processing factory in Tibet. Five townships were chosen in each county based on 5 different directions of east, south, west, north and center. If the monitoring county has less than five townships, then all of the townships were sampled. In each township, four villages were selected withrandom sampling and importance sampling. In each township, 15 households were selected for salt collection. Results A batch of 15 salt samples in a salt processing plant were tested, and all of them were qualified with salt iodine(34.6±1.58) mg/kg. A total of 21 107 edible salt samples were tested, and 11 203 of them were qualified iodized salt. These results meant that the provincial iodized salt coverage rate was 53.08%. Shannan iodized salt coverage rate was 94.31% (3395/3600) which was the highest in Tibet. Those of Nagqu, Changdu, Ngari were lower, they were 29.84% (897/3006), 24.94% (823/3300) and 17.08% (205/1200), respectively. Conclusions The quality of iodized salt in Tibet is up to the national standard, but the coverage rate of iodized salt is very low.We suggest that the strategy should be carried out according to the national overall program strategy and supplement iodized oil capsule for special groups.

OBJECTIVETo analyze the complications of bipolar hemiarthroplasty for the treatment of intertrochanteric fractures.

METHODSFrom Jan. 2004 to Dec. 2007, 62 patients over 80 years old with unstable interthochanteric fracture were treated with bipolar hemiarthroplasty, included 34 males and 28 females with an average age of 86.3 years ranging from 81 to 97 years. According to the Evans classification, there were 29 cases of Evans III, 26 of Evans IV and 7 of Evans V. The systemic and operation related complications were investigated.

RESULTSAmong all the cases, 59 were followed up in outpatient department for 24 to 70 months (33 months on average). Systemic complications were found in 19 cases with no death during preoperative period and 5 deaths after leaving hospital. Operation related complications were found in 9 cases, included 3 cases of thigh pain, 1 iatrogenic fracture of proximal femur, 2 hip dislocations, 2 delayed union of fractures and 1 superficial infection. There were no aseptic loosening, peri-prosthetic infections,ectopic ossification or injuries of nerves and vessels.

CONCLUSIONBipolar hemiarthroplasty is indicated for patients over 80 years old with intertrochanteric fracture, thus the organic or systemic malfunctions should be corrected during perioperative period. Meanwhile, retaining of lesser trochanter and reconstruction of calcar femorale are important for improving periprosthetic biomechanics and reducing local complications.

Aged, 80 and over ; Arthroplasty ; adverse effects ; Female ; Follow-Up Studies ; Hip Fractures ; diagnostic imaging ; physiopathology ; surgery ; Humans ; Male ; Postoperative Complications ; Recovery of Function ; Tomography, X-Ray Computed

Objeetive : The epidemiological characteristics of confirmed cases of the 2019 novel coronavirus diseases (COVID-19) which was reported in Wenzhou by February 28,2020 were described and analyzed to provide evidence for prevention and control measures in next stage. Methods: The field investigation method was employed on all suspected and confirmed cases. The data of time,spatial and population distribution, source of infection and detective routes of all confirmed cases of COVID-19 in Wenzhou were collected and extracted from the reports of field investigation and the National Diseases Prevention and Control Information System. Results: As of February 20,2020,504 confirmed cases of COVID-19 were diagnosed in Wenzhou, which included one death case. Among all 13 counties, the highest number of confirmed cases were distributed in Yueqing,Ruian and Lucheng. Among 504 cases, the male to female ratio was reported as 1.14:1. The youngest case was 2 years old while the oldest case was 93 years old. Most of the confirmed cases were among people between the ages of 25 and 64 years old. The most common occupation of the confirmed cases was businessman,which accounts for 29.76% (150 cases). Clinical symptoms of the first confirmed case of COVID-19 in Wenzhou occurred on January 4, 2020. Approximately 70% of cases had the first symptoms from January 19 to February 1. The daily number of new cases on January 24 and January 26 were the highest while the number of new cases per day declined significantly after February 2, and there were no new cases after February 14. Among all confirmed cases, the infection source of 61.71% cases were related to Wuhan. As of February 20, 2020, a total of 79 clusters were reported in Wenzhou, which involved 330 cases. Approximately 30% of confirmed cases came to hospitals unaccompanied by community staffs, and cases which confirmed after February 13 were basically found in central isolation locations or by grid checkers. Conclusions The main reason for the high case numbers of COVID-19 in Wenzhou, in Zhejiang province, was the high number of wenzhou citizens who work in Wuhan. Imported cases account for the most confirmed cases during the early period, while mainly confirmed cases were local cases during the middle and late periods. The outcomes suggest the prevention and control measures employed by Wenzhou government, which included closed-loop control and quantitative control, have achieved remarkable results and the epidemic of COVID-19 in Wenzhou has been initially controlled.

Objective To investigate the relationship between the expression of long non-coding RNA HOXA-AS2(lncRNA HOXA-AS2),long non-coding RNA FOXD2-AS1(lncRNA FOXD2-AS1),and long non-coding RNA CRNDE(lncRNA CRNDE)in endometrial carcinoma and the clinical pathological character-istics and prognosis of patients.Methods Collect samples of endometrial carcinoma cancer tissues and adja-cent tissues excised during surgery from 119 endometrial carcinoma patients admitted to a hospital from Octo-ber 2017 to February 2020.The relative expression levels of HOXA-AS2,FOXD2-AS1 and CRNDE in tissues were retrospectively analyzed,as well as their relationship with clinicopathological features and 3-year survival rate of patients.Results The relative expression levels of HOXA-AS2,FOXD2-AS1 and CRNDE in cancer tissues of endometrial carcinoma patients were higher than those in adjacent tissues,with statistical signifi-cance(P＜0.05).The relative expression levels of HOXA-AS2,FOXD2-AS1 and CRNDE in cancer tissues of endometrial carcinoma patients were positively correlated(rHOXA-As2 vs.FOXD2-AS1=0.384,P=0.001;rHoXA-AS2 vs.CRNDE=0.576,P＜0.001;rFoXD2-AS1 vs.CRNDE=0.326,P=0.003).In the HOXA-AS2,FOXD2-AS1 and CRNDE high expression group,the proportion of patients with international federation of gynecology and ob-stetrics(FIGO)stage Ⅲ+Ⅳ,lymph node metastasis,deep infiltration and low differentiation was higher than that in the low expression group,with statistical significance(P＜0.05).The 3-year survival rate of low HOXA-AS2 expression group in endometrial cancer patients(52/60,86.67％)was higher than that of high HOXA-AS2 expression group(40/59,67.79％),the difference was statistically significant(x2=6.039,P＜0.05).The 3-year survival rate of patients with endometrial cancer with low FOXD2-AS1 expression group(53/59,89.83％)was higher than that of patients with endometrial cancer with high FOXD2-AS1 expression group(39/60,65.00％),and the difference was statistically significant(x2=10.456,P＜0.05).The 3-year sur-vival rate of low CRNDE expression group in endometrial cancer patients(51/60,85.00％)was higher than that of high CRNDE expression group(41/59,69.49％),and the difference was statistically significant(x2=4.079,P＜0.05).HOXA-AS2,FOXD2-AS1,and CRNDE were risk factors for death in endometrial carcinoma patients(P＜0.05).Conclusion The expression of HOXA-AS2,FOXD2-AS1,and CRNDE in endometrial carcinoma cancer tissue is closely related to the clinical pathological characteristics and prognosis of patients.

Objective:To evaluate the effect of remimazolam on hemodynamics when used for anesthesia induction in patients undergoing coronary artery bypass grafting (CABG).Methods:Sixty patients of either sex, aged 18-64 yr, of American Society of Anesthesiologists Physical Status classification Ⅱ-Ⅲ, with ejection fraction>35%, cardiac index (CI) >2.0 L·min -1·m -2 and body mass index <28 kg/m 2, undergoing CABG under general anesthesia, were divided into 2 groups ( n=30 each)using a random number table method: remazolam group (group R) and midazolam group (group M). Group R received intravenous remimazolam 0.08 mg/kg, and group M received intravenous midazolam 0.05 mg/kg, and both groups used sufentanil, etomidate, and rocuronium bromide for anesthesia induction to maintain the BIS value at 40-65. Before anesthesia induction, at 1-2 min after intravenous injection of remimazolam/midazolam-sufantanil-etomidate, immediately before tracheal incubation, at 30 s-15 min after tracheal intubation, and immediately before skin incision, blood pressure, heart rate, mean arterial pressure, CI, stroke volume index, systemic vascular resistance index, and left heart systolic index were measured, and vasoactive drugs were used to maintain the stability of vital signs and to record the cardiovascular events. Results:Compared with group M, the incidence of hypotension and usage rate of vasoactive drugs were significantly decreased, and the incidence of decrease in CI and stroke volume index by more than 20% and changes in left heart systolic index were decreased in group R( P<0.05). Conclusions:Remimazolam can reduce the disturbance of hemodynamics to a certain extent when used for anesthesia induction as compared with midazolam in patients undergoing CABG.

OBJECTIVETo study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).

METHODSThe Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR.

RESULTSThe TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA.

CONCLUSIONA Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; physiology ; Humans ; Tetracycline ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; genetics ; Virus Replication