Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective: This paper develops a methodology for steady and orderly expansion of the designated medical insurance drugstores. Methods:With the help of grid management ideology, the paper uses ArcGIS and its function modules to define and visualize the scope of the medical insurance drugs service with the guidance of fairness and efficiency through analyzing the basic data including population distribution, the physical distribution of drug-stores and soon. Results:In order to figure out the procedure of the methodology,the key problems were all solved, including defining and visualizing the scope of the medical insurance drugs service area,,checking and ratifying the scope that need to supplement or increase the medical insurance drugstores, making measures for the annual imple-mentation and confirming a selection principle for designated medical insurance drugstores. Conclusion:The Method-ology guided by fairness and efficiency of the medicare designated pharmacy layout planning is practical and feasible, which can probably provide a reference for increasing the designated medical insurance drugstores steadily and orderly.

OBJECTIVETo investigate antimicrobial resistance of Klebsiella pneumoniae and Pseudomonas aeruginosa isolates in fecal samples from rat-like animals.

METHODSRat-like animals were captured using cages around a hospital and the neighboring residential area between March and October, 2015. K. pneumoniae and P. aeruginosa were isolated from the fecal samples of the captured animals. Antimicrobial susceptibility test was performed according to the guidelines of Clinical and Laboratory Standards Institute (2014).

RESULTSA total of 329 rat-like animals were captured, including 205 Suncus murinus, 111 Rattus norvegicus, 5 Rattus flavipectus and 8 Mus musculus. The positivity rates of K. pneumoniae and P. aeruginosa were 78.4% and 34.7% in the fecal samples from the captured animals, respectively. K. pneumoniae isolates from Suncus murinus showed a high resistance to ampicillin, cephazolin, nitrofurantoin, piperacillin and cefotaxime (with resistance rates of 100%, 51.2%, 44.2%, 37.2%, and 23.3%, respectively), and K. pneumoniae isolates from Rattus spp. showed a similar drug-resistance profile. The prevalence rates of multidrug resistance and ESBLs were 40.9% and 10.7%, respectively. P. aeruginosa from both Suncus murinus and Rattus spp. exhibited the highest resistance rates to aztreonam (12.4% and 16.0%, respectively), followed by penicillins and fluoroquinolones. P. aeruginosa isolates were susceptible to cephems, aminoglycosides and carbapenems (with resistance rates below 5%).

CONCLUSIONK. pneumoniae and P. aeruginosa isolated from rat-like animals showed drug-resistance profiles similar to those of the strains isolated from clinical patients, suggesting that the possible transmission of K. pneumoniae and P. aeruginosa between rat-like animals and human beings.

Animals ; Anti-Bacterial Agents ; pharmacology ; Carbapenems ; pharmacology ; Drug Resistance, Bacterial ; Fluoroquinolones ; pharmacology ; Humans ; Klebsiella pneumoniae ; drug effects ; isolation & purification ; Mice ; Murinae ; microbiology ; Pseudomonas aeruginosa ; drug effects ; isolation & purification ; Rats

Antihypertensive Agents ; therapeutic use ; Blood Pressure ; physiology ; China ; Consensus ; Humans ; Hypertension ; diagnosis ; drug therapy ; epidemiology

This article shared opinions on how to effective educate clinical type postgraduates in department of neurosurgery based on the teaching models in the first affiliated hospital of Chongqing medical university. In the proposed training program,medical ethics education and comprehensive training including clinical thinking and practical skills training should be emphasized.Scientific inquisitiveness of the neurological research and the writing and oral communication in English should also be promoted.This training program will be fully committed to produce highly qualified,ethical and caring neurosurgeons for future needs of the society.

Objective To study the efficiacy and safety of transfacet approach decompression to treat thoracic spinal stenosis caused by anterior compression.Methods Thirty-three patients with thoracic spinal stenosis caused by anterior compression were treated in our institution from April 2005 to April 2009.Nineteen patients with more than 12 months follow-up were included in this study.Among of them,10 were male and 9 were female,with the age ranged from 33 to 77 years(mean,55.9 years).The causes of compression for spinal stenosis included ossification of posterior longitudinal ligament(OPLL)in 5 cases,thoracic disc herniation(TDH)in 11 cases,OPLL with ossification of ligamentum flavum(OLF)in 2 cases and TDH with OLF in 1 case.All patients underwent anterior decompression via a transfacet approach combined with anterior fusion and posterior fixation.The modified Japanese Orthopaedic Association(JOA)score system and Nurick Myelopathy grade were used to evaluate the outcomes.Results The operation time ranged from 180 to 480 min,with an average of 299.5 min and the blood loss was varied from 250 to 2200 ml,with an average of 918.5 ml.Among 7 cases with OPLL(including combined with OLF),2 patients developed neurologic deterioration and 1 patient developed cerebrospinal fluid leakage.There were no neurologic deterioration,cerebrospinal fluid leakage and other complications occurred in 12 cases with TDH(including combined with OLF).The follow-up ranged from 12 to 54 months(mean,28.6 months).The preoperative JOA score ranged from 2 to 11(mean,6.3).The JOA score in the last follow-up ranged from 5 to 11(mean,8.6).According to Nurick Myelopathy grade,the preoperative grade was 0 in 2 cases,1 in 2,2 in 4,3 in 5,4 in 2,and 5 in 4.The number of postoperative grade was 6,6,3,3,1 and 0 respectively.Conclusion Satisfactory decompression could be achieved by using transfacet approach for thoracic spinal stenosis caused by anterior compression.The approach is a safe and promising alternative for thoracic spinal anterior decompression.

OBJECTIVETo evaluate the cellular and humoral immunity effect of 10 µg and 20 µg recombinant Chinese hamster ovary (CHO) cell hepatitis B vaccine in adults by randomized double-blind controlled trials.

METHODA total of 642 adults aged 18 - 45 years old, non-vaccinated against hepatitis B, and hepatitis B five blood indicators negative were selected as the study subjects. The study subjects were randomly divided into two groups and each group had 321 subjects. The subjects were given 10 µg and 20 µg recombinant CHO hepatitis B vaccination respectively by 0, 1st, 6th month schedule. Blood sample was collected from each study subject one month after the second dose vaccination. The anti-HBs level was detected by Abbott chemiluminescence detection method (I2000) to evaluate humoral immunity status. Of all the study objects, 153 cases were randomly selected by the Excel random function. Their blood samples were collected and Lymphocyte were separated to detect the IL-4 and IFN-γ levels in vitro by enzyme-linked immunospot (ELISPOT) method to evaluate the cellular immunity status.

RESULTSThe anti-HBs seroconversion rates in 10 µg and 20 µg dose group were 88.8% (285/321) and 95.3% (306/321) respectively, and 95%CI were 85.4% - 92.2% and 93.0% - 97.6% respectively. The spot forming cell (SFC) of IL-4 of the 20 µg-dose group (x(-) = 20.31) were significantly higher than the 10 µg-dose group (x(-) = 8.19, t = 3.27, P < 0.01). With the increasing of anti-HBs titer, the SFC of IL-4 also went up significantly. There was a positive correlation between SFC of IL-4 and anti-HBs (Spearman correlation coefficient = 0.538, P < 0.0001). No significant difference was found for IFN-γ SFC in two groups (10 µg group: x(-) = 1.49; 20 µg group: x(-) = 0.86; t = 1.83, P > 0.05).

CONCLUSIONThe humoral and cellular immune effects of 20 µg recombinant CHO hepatitis B vaccine are better than that of the 10 µg recombinant CHO hepatitis B vaccine.20 µg recombinant CHO hepatitis B vaccine should be chosen as the adult's hepatitis B prevention vaccine.

Adolescent ; Adult ; Animals ; Antibody Formation ; CHO Cells ; Cricetinae ; Cricetulus ; Double-Blind Method ; Female ; Hepatitis B ; prevention & control ; Hepatitis B Antibodies ; blood ; immunology ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Humans ; Immunity, Cellular ; immunology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo study the effects of L-arginine (L-Arg) on human colon carcinoma cell line LS174 through NO pathway and the mechanism.

METHODSLS174 cells were cultured in the presence of L-Arg at different concentrations for different time. MTT assay was employed to evaluate the cell proliferation, and the cell morphological changes were observed by optical and electron microscopy. The apoptosis of L-Arg-treated cells was observed by the Annexin V/PI staining. The expression levels of VEGF, COX-2, p53, bcl-2 and bax in the cells were determined using Western blotting and immunocytochemical staining.

RESULTSL-Arg promoted the growth of LS174 cells at a low concentration (0.125 mmol/L) and inhibited the cell growth at higher concentrations (0.5, 2, 8, and 32 mmol/L). Under electron microscope, ultrastructual changes in the cytoplasm and nuclei of LS174 cells were observed 48 h after exposure to L-Arg. Some of the cells showed morphological changes characteristic of cell apoptosis such as cell size reduction, condensation and margination of the nuclear chromatin. Low concentration of L-Arg resulted in a cell apoptosis rate of 2.29%, as compared with the rate of 2.84% in the control group; at higher concentrations, L-Arg caused significantly increased cell apoptosis rates to 26.88%, 28.95%, 63.36%, and 84.51%, respectively. In cells treated with a low concentration of L-Arg, the expressions of VEGF and COX-2 were increased as compared with those in the control cells; higher concentrations of L-Arg obviously decreased the expressions of p53 and bcl-2 and increased the expression of bax.

CONCLUSIONLow-concentration L-Arg can promote the growth of LS174 cells probably by up-regulating VEGF and COX-2 protein under the influence of NO, the metabolite of L-Arg. The inhibitory effect of high concentrations of L-Arg is probably mediated by inducing cell apoptosis via NO. The mechanism of L-Arg- induced cell apoptosis may be related to the up-regulation of bax protein and the down-regulation of p53 and bcl-2 proteins.

Apoptosis ; drug effects ; Arginine ; pharmacology ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Cyclooxygenase 2 ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Nitric Oxide ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

Objective To evaluate the immuno-effect and related influencing factors on 10 μg and 20 μg Chinese hamster ovary (CHO) cell hepatitis B vaccine, using the randomized double-blind controlled trials in adult population. Methods A total of 642 adults aged 18-45 years old, non-vaccinated against hepatitis B, and negative on five blood indicators for hepatitis B, were selected as the study objects from four districts in Beijing. The study objects were randomly divided into two groups, and then accepted 10 tg and 20 μg recombinant CHO hepatitis B vaccination by 0-1-6 month schedule. Influencing factors were investigated by means of questionnaire. Blood samples were collected one month after the third dose of vaccination. Anti-HBs level was detected by Abott chemiluminescence detection method. For the anti-HBs negative person, fluorescent quantitative PCR method was used to find out if the person had been infected with HBV. Logistic regression analysis was used to find out the influencing factors of anti-HBs seroconversion on every studied subject. Results The anti-HBs seroconversion rates on 10 μg and 20 μg dose groups were 88.8%(95%CI: 85.4%-92.2%) and 95.3%(95%CI: 93.0%-97.6%)respectively. Taking the anti-HBs level＜100 mIU/ml as the low/non-response standard, the low response and non-response rates were 34.3% and 17.4% respectively. The geometric mean titers(GMT)of anti-HBs were 173.42 mIU/ml for the 10 μg dose group and 588.51 mIU/ml for the 20 μg dose group. Data from the Multivariate analysis showed that: diabetes, spouses infected with hepatitis B virus and old age were unfavorable factors for anti-HBs Seroconversion. 20 μg dose of the vaccine was conducive to seroconversion.Conclusion 20 μg CHO hepatitis B vaccine seemed better than 10 μg CHO hepatitis B vaccine while many factors need to be taken into account for evaluation on hepatitis B vaccines.

Objective: To determine the expression of human telomerase reverse transcriptase (hTERT) and P53 in thyroid carcinoma and its relationship with development and prognosis of the carcinoma. Methods: Totally 90 cases of thyroid specimens (60 thyroid carcinomas, 10 thyroid adenomas, 10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results: Positive immunoreactivity of hTERT and P53 was higher in thyroid carcinoma (P<0.05). The positive rates of hTERT and P53 were higher in undifferentiated carcinomas, carcinomas with lymph nodes metastasis or at stage III + IV than in well-differentiated carcinomas, carcinomas without lymph nodes metastasis or at stage I + II (P<0.05). The expression of hTERT was significantly related with that of P53 (P<0.05). Conclusion: Over-expressed hTERT and P53 may be related to the carcinogenesis and progression of thyroid carcinoma and hTERT expression is related to P53 protein. Examination of expression of hTERT and P53 proteins may be helpful to judge the thyroid cancer's behavior and prognosis.