Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective: To observe the clinical effect of traditional Chinese medicine enema combined with rhubarb acupoint in the treatment of severe acute pancreatitis(SAP) complicated with paralytic ileus. Methods: A total of 60 SAP patients complicated with paralytic ileus treated in the First People's Hospital of Huzhou from June 2015 to June 2017 were selected and randomly divided into two groups by random lottery method, with 30 cases in each group.The two groups were treated with routine anti-infection and rehydration.The control group was treated with traditional Chinese medicine enema combined with placebo, and the observation group was treated with traditional Chinese medicine enema combined with rhubarb on Shenque acupoint, and the two groups were treated for 7d.The recovery time of blood amylase, bowel sounds recovery time, first self defecation time, local complications and mortality were observed in the two groups. Results: The recovery time of blood amylase, the recovery time of intestinal sound and the time of the first defecation were (8.87±2.30)d, (2.88±1.09)d and (3.51±1.32)d, respectively, which were significantly shorter than those of the control group [(10.29±2.01)d, (3.67±1.30)d and (4.57±1.41)d], the differences were statistically significant(t=-3.601, -3.607, -4.251, all P<0.001). The incidence rate of local complications in the observation group was 6.7%(2/30), which was significantly lower than 36.7%(11/30) in the control group(χ²=16.875, P<0.001), and there was no statistically significant difference in the mortality rate between the two groups(χ²=3.333, P=0.068). Conclusion The application of Chinese medicine enema combined with the acupoint of rhubarb in the treatment of SAP patients complicated with paralytic ileus can effectively promote the recovery of intestinal peristalsis, shorten the duration of paralytic intestinal obstruction, reduce the complications and improve the clinical effect.

Objective To improve the efficiency of blood collection in hemodialysis patients by inventing and applying new blood collection needles.Methods One hundred and eighty cases of hemodialysis patients were randomly divided into control group 1(CG1),control group 2(CG2),experimental group(EG). Comparison of the three groups in time of blood collection,the number of operation that had the risk of acupuncture injury and the number of the blood samples that had been contaminated. Results There were statistically significant difference (P<0.05)in three groups.The number of times of blood collection in the number of operations that had the risk of acupuncture injury,and the number of the blood sample that had been contaminated. CG1 had the longest blood collection time(12.55 min),EG had the shortest blood collection time(5.09 min);the risk of acupuncture injury was the highest in CG2 and the lowest in the EG.The number of contamination of blood samples and the amount of inaccurate sample in CG1 were the highest,and the lowest in the EG. Conclusions The working efficiency of transfer type anti-acupuncture needle(TTAN)during blood sample collection in hemodialysis patients is signifi-cantly better than that of traditional blood collection method,and it is helpful to reduce the risk of acupuncture injury and the risk of contamination in blood samples,which is worthy of promotion.

Objective To analyze the related risk factors of functional intestinal obstruction in general surgical patients and to discuss the feasible nursing and prevention measures. Methods The clinical data of totally 658 patients after abdominal surgery who received open abdominal surgery in the Department of General Surgery of the First People's Hospital of Huzhou in Zhejiang Province from January 2015 to January 2017 were retrospectively analyzed. These patients were divided into the intestinal obstruction group and the non-intestinal obstruction group based on the occurrence of postoperative functional intestinal obstruction. The single factor analysis was then conducted over the age, gender, operation time, anesthesia, postoperative analgesic use (opioids and non-opioids), abdominal operation history, smoking history, alcohol drinking history, diabetes mellitus history and history of hypertension; and Logistic regression analysis was also performed on the influencing factors to determine the impact of functional intestinal obstruction in patients who received open abdominal surgery. Results Totally 47 out of the 658 patients receiving open abdominal surgery developed functional intestinal obstruction, with an incidence of 7.14%. The univariate analysis showed that there were statistically significant differences between the two groups in gender, age, operation time, postoperative analgesic use, history of abdominal surgery and history of diabetes mellitus (P<0.05). According to the multivariate Logistic regression analysis, gender, age, operation time, history of abdominal surgery and history of diabetes were the risk factors of postoperative functional intestinal obstruction (P<0.01). Conclusions The relevant risk factors for postoperative functional intestinal obstruction in general surgical patients after abdominal surgery include gender, age, operation time, history of abdominal surgery and diabetes mellitus. Corresponding nursing and prevention measures should be taken according to the relevant risk factors of patients.

OBJECTIVE:To analysis the volatile components in wild strains of Rabdosia rubescensin in Jiyuan area. METH-ODS:Headspace solid phase microextraction (HS-SPME) was conducted to extract the volatile components in 43 wild species of R. rubescens,GC-MS was conducted to identify the components,and peak area normalization method was conducted to calculate the relative content of the components. RESULTS:The variety and relative contents of the volatility components in 43 wild species had obvious differences and the ranges of species number and relative content was from 0 to 11 and from 0 to 84.03% respectively. Caryophyllene,caryophyllene oxide,dihydroactinidiolide,phytone and patchouli alcohol were main components which existed in a variety of different strains of volatile components of R. rubescens,and were mainly composed of terpenes,ketones,alcohols and es-ters. In addition,fatty acids such as palmitic acid,oleic acid and linoleic acid were contained in a few individual varieties. CON-CLUSIONS:The internal quality in 43 wild species of R. rubescens has certain differences.

Objective To know the progress of research in oral care of orotracheal intubated patients in China and to analyze the existing problems as well as provide the reference for further study.Methods Databases of CNKI,Wanfang,VIP and CBM were searched for literature on oral care for the orotracheal intubated patients by the end of December 2013,and the methods of bibliometrics were used to analyze the research results.Results Three hundred and nine articles that met the inclusion and exclusion criteria from 1997 to 2013 were collected.These papers were mainly included in 22 kinds of nursing journals.The methods of oral care for orotracheal intubated patients could be classified into five types:rinse and swabbing (95 papers),traditional cotton(cotton swab) cleaning or gauze swabbing (58 papers),toothbrushing (54 papers),oral rinse (51 papers),laryngoscope assistance (7 papers).With 54 articles using traditional cotton balls as oral care tool,55 papers using toothbrush,including children toothbrush of soft bristle (29 papers),ordinary toothbrush (14 papers) and electric toothbrush (12 papers).Oral care solution mainly included normal saline(49 papers),traditional Chinese medicine (36 papers),chlorhexidine(36 papers),antibiotics (12 papers),povidone iodine (17 papers),sodium bicarbonate (9 papers),etc.Conclusions The oral care of orotracheal intubated patients has attracted the attention of researchers,but the use of oral care methods,the frequency in oral care and oral care tools still owe specification.More attention should be paid to strengthen the training of nursing staff,to improve the scientific research ability of scientific research workers and to carry out large sample and high quality trials.An oral care guideline that fits into our national conditions should be built in an effort to improve the level of oral care in critical patients in our country.

Objective To observe the effect of rehabilitation gymnastics with music on non-incisional pain of gynecological patients after laparoscopic operation.Methods 192 gynecological patients undergone laparoscopic operation were divided into the music group (66 cases, adopted rehabilitation gymnastics matching music after operation), non-music group (64 cases, adopted rehabilitation gymnastics) and routine group (62 cases, adopted routine nursing without rehabilitation gymnastics or music).Results The patients of the music group got pain eased more obviously than those of the non-music group and rule group ( P<0.05).Conclusion The rehabilitation gymnastics with music can relieve non-incisional pain after gynecological laparoscopic operation, and improve anus exhausting.