BACKGROUND:As a novel growth factor, human intestinal trefoil factor (TFF3) can promote cel growth and migration, and increase cel resistance to apoptosis, and it plays a great role in maintaining the mucosa integrity, mucosa protection and repairing the injured mucosa, also it has been closely related to the tumor growth and progression. With the function of mucosa repair, and as the tumor biomarker, TFF3 has a promising clinical application, but its definite interacting protein and molecular mechanism is stil unclear. OBJECTIVE:To construct and express the TFF3 recombinant protein with the tandem tag of StrepII-6×His in the target cel s for further purifying its interaction protein in the native condition based on the tandem affinity purification technique. METHODS:The DNA sequence for the tag (StrepII-TEV-6×His) and TFF3 as template was got by chemical synthesis and PCR amplification respectively. They were fused by the restriction enzyme XbaI site, and the tag sequence was located at the C terminus of TFF3 protein. TFF3-tag fusion gene was cloned into the pCDNA3.0 using EcoRI+HindIII, thus the TFF3-tag expressing vector pTFF3-C-StH was constructed and transfected transiently into gastric cel AGS by lipofectin. The recombinant TFF3-tag protein was expressed and detected by western blot assay. RESULTS AND CONCLUSION:The expressing vector pTFF3-C-StH for tandem affinity purification was constructed successful y, and was confirmed further by restriction enzyme analysis and sequenced. The recombinant TFF3-C-StH protein of TFF3-tag was expressed in the AGS cel , and showed specific antigenicity by western blot assay. Thus this work provides experimental base for further purification of the TFF3 interacting proteins.

Objective To investigate the role of platelet indices in the differentiation of thrombocytopenia caused by bone marrow diseases and thrombocytopenia caused by non-marrow diseases.Methods Mean platelet volume(MPV),platelet crit(PCT)and platelet size deviation width(PDW)of 76 othromboeytopenia(TP)patients,66 immune thrombocytopenia purpura(ITP)patients,8 secondary thrombocytocritpenia purpura(STP)patients,8 acquired pure megakaryocytic thrombocytopenia purpura(AATP)patients,140 malignant hematological diseases(MHD)(including acute leukemia,aplastic anemia and myelodysplastic syndromes)patients and 100 healthy people were respectively determined by blood cell analyzer Cell-Dyn 3700.Result Compared with control group,the MPV of the patients with MHD significantly decreased,and then the PDW of them increased.For patients with STP,the MVP and PDW markedly increased.The MVP of patients with AATP was normal,and PDW significantly increased.The MPV,PCT and PDW of ITP and TP showed marked difference(P＜0.01).In contrast with the control group,the MPV,PCT and the PDW of patients with hemopathy and patients with ITP were remarkably different(P＜0.01).Compared with the marrow TP and the non-marrow TP or control group,MPV and PDW showed significant difference(P＜0.01).During the effective treatment of patients with ITP,PLT increased and then MPV and PDW decreased.Conclusion The level changes of MPV and PDW,in particular MPV can primarily judge the cause of thrombocytopenia (MPV＜reference value as caused by bone marrow diseases but MPV＞reference value as caused by bone marrow diseases).In addition,during the effective treatment of patients with ITP,with changes of MPV and PDW present 5 days earlier than that of PLT,may used to observe the therapeutic effect.Myelodysplastic syndromedynamic observation of the platelet parameter helps for the differential diagnosis,judgment of the states of the diseases and observation of the effectiveness of therapy of ITP.

Objective To study the clinical efficacy of the injection Salvia (freeze-dried, abbr: Salvia powder needle), with fallopian tube recanalization on the treatment of oviduct obstructive infertility. Methods One hundred and ninety-five patients of oviduct obstructive infertility were enrolled in this study and divided into drug group and control group. Salpingography with modified catheter, recanalization and perfusion operation were performed in both two groups. Salvia powder, gentamicin, α-ACT and dexamethasone were injected into test group, while Gentamicin, α-ACT and dexamethasone were injected into control group. Recanalization rate of fallopian tube, rate of intrauterine gestation and rate of ectopic pregancancy were measured and compared between two groups. Results Twelve months after operation, recanalization of fallopian tube of test group and control group was 94.39% and 85.87%, respectively. Twenty-four months after operation, intrauterine gestation rate of test group and control group was 50.00% and 35.71%, respectively. Conclusion Salvia powder combined with modified recanalization operation is helpful to improve the rate of intrauterine gestation.

Objective To establish a novel method to screen for anti-varicella-zoster virus (VZV) compounds with our previously generated reporter cell line for VZV, MV9G. MethodsMV9G cells were directly infected with cell-free virus of Oka vaccine strain (vOka) for 2 hours( CFV direct-infection) or cocultured with vOka-infected MeWo cells containing cell-associated virus for 48 hours (CAV co-culture) to promote expression of the reporter gene firefly luciferase. Antiviral compounds including heparin, mannose-6-phosphate( M-6-P), acyclovir( ACV ), resveratrol and roscovitine were added in the medium before or after the virus infection. Inhibitory effects( IC50 ) of the antiviral compounds were analyzed by comparing firefly luciferase activities of MV9G cells in the presence of antiviral compounds with those in the absence. Results Antiviral compounds inhibited luciferase activities of MV9G cells activated by CFV direct-infection and/or CAV co-culture in different levels. The reductions of luciferase activities statistically correlated with those of viral foci shown by immunostaining with a monoclonal antibody against VZV immediate early 62 antigens (IE62) in controls. Among these compounds, heparin, M-6-P, and 2.5 μmol/L of roscovitine inhibited CFV-activated more strongly than CAV-activated luciferase activities, whereas ACV and resveratrol inhibited CAV-activated more strongly than CFV-activated luciferase activities. Cell-associated ACV-resistant strains,Kanno and rOka YSR, activated luciferase activities of MV9G cells, too. However, the inhibitory concentrations (IC50) of ACV to the ACV-resistant strains were much higher than those to the ACV-sensitive strains,pOka and CaGu. ConclusionThe CFV direct-infection and CAV co-culture assays were useful to screen for antiviral compounds targeting the early and late phases of VZV infection, respectively. The VZV reporter cell-based assays may provide a simple, rapid, sensitive, and high-throughput method to screen for anti-VZV compounds.

Objective To explore the potential association of HLA-A alleles and genetic susceptibility with systemic lupus erythematosus (SLE). Methods Polymerase chain reaction-sequence specific primer (PCR-SSP) was used to analyze the distribution of HLA-A alleles among 106 patients with systemic lupus erythematosus and 122 healthy persons. Results Nineteen out of twenty-four kinds of HLA-A alleles were found from the specimens, including 18 kinds in SLE specimens, and 15 kinds in control specimens. Among them, HLA-A*11 allele was positively associated with SLE (RR = 2.4380, EF = 0.1502, ?2 = 12.2440, P = 0.0005, Pc = 0.0095). For A*01 and A*24, although the P values were less than 0.05, the Pc values were more than 0.05 (0.9462 or 0.2356, respectively). Conclusions The results indicate that HLA-A*11 may be the susceptible allele or may be closely linked with the susceptible genes in Chinese SLE patients.

Spider dragline silk is one of most perfect fibrous proteins in nature. As biomaterials, it has a wide application in tissue engineering due to its unique mechanical properties, good biocompatibility, slow degradation. In this paper, based on the highly repetitive sequence of spider dragline silk and with the introduced RGD peptide codons which involve cell adhesion, the DNA monomer sequence encoding RGD-spider dragline silk was synthesized, and then was used to construct the multimers by the strategy of "head to tail"; the multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLyS, were transformed the expression of recombinant protein was induced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 35KD and 60KD respectively in agreement with the desired. Western assay was used for determining the specification of products. Further, the purification process was groped for the producing of large quantity of synthetic proteins through high density fermentation.
Amino Acid Sequence ; Animals ; Base Sequence ; Biocompatible Materials ; Cloning, Molecular ; Fibroins ; biosynthesis ; genetics ; isolation & purification ; Molecular Sequence Data ; Oligopeptides ; biosynthesis ; isolation & purification ; Polymers ; isolation & purification ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Spiders

Objective: To compare the effect of scalp acupuncture and scalp acupuncture plus acupuncture exercise therapy (AET) on walking ability in children with spastic cerebral palsy (CP). Methods: A total of 60 spastic CP children with gross motor function classification system (GMFCS) grades Ⅰ-Ⅲ were divided into a control group and an observation group by the random number table method, with 30 cases in each group. Both groups were treated with the same conventional rehabilitation and scalp acupuncture therapy for CP. The control group received conventional rehabilitation first and then scalp acupuncture. The observation group received AET, which was to receive the conventional rehabilitation and scalp acupuncture simultaneously. Before and after treatment, the clinical efficacy was evaluated by the modified Ashworth scale (MAS) score, scores of dimensions D and E of the gross motor function measure (GMFM) scale, walking speed, and walking distance. Results: During treatment, there were 2 dropouts in the observation group. After 3 courses of treatment, the MAS scores in both the control group and observation group decreased compared with the same group before treatment (P<0.05), and the scores of dimensions D and E of the GMFM, walking speed, and walking distance were increased (P<0.05); the between-group comparison showed that the MAS score in the observation group was lower than that in the control group (P<0.05), and the scores of dimensions D and E of the GMFM, walking speed, and walking distance in the observation group were higher or longer than those in the control group (P<0.05). Conclusion: W ith the same treatments, scalp acupuncture combined with AET is superior to the conventional scalp acupuncture method in reducing lower-limb muscle tone, improving standing balance ability, and walking stability in children with spastic CP.

Objective:To predict the mechanism of Danggui Buxue Decoction for anti-myocardial ischemia-reperfusion injury and "treating different diseases with the same method" in ischemic stroke based on network pharmacology and molecular docking.Methods:The active components and targets of Danggui Buxue Decoction were screened by retrieving the database of TCMSP and literature; the corresponding targets of myocardial ischemia-reperfusion injury and ischemic stroke were found by OMIM and GeneCards database; the intersection targets of Danggui Buxue Decoction and disease were obtained by using Venny diagram, and the common target network and protein-protein interaction network were constructed by Cytoscape 3.7.1 software and STRING database. The GO and KEGG pathways were enriched by David Database, and the Bio GPS database was used to obtain the tissue distribution information of the key targets. The molecular docking technology was used to verify the results.Results:There were 21 active components in Danggui Buxue Decoction, 181 effective targets and 93 cross targets with diseases. The key components were quercetin, Kaempferol, β-sitosterol, formononetin and isorhamnetin. The key targets were AKT1, TNF, IL6, IL-1β and VEGFA. The enrichment results showed that the main action pathways were fluid shear force and arteriosclerosis, lipid and arteriosclerosis, AGE-RAGE signal pathway in diabetic complications, and the core targets were mainly located in the medullary cells, dendritic cell, smooth muscle, prostate, thyroid and other tissues. The results of molecular docking showed that quercetin had the best binding effect to IL-1β, while isorhamnetin had the best binding effect to IL-1β.Conclusion:Danggui Buxue Decoction is against myocardial ischemia-reperfusion injury and ischemic stroke through hemodynamics, lipid metabolism, inflammatory reaction, oxidative stress, immune reaction and cell apoptosis, plays the role of "treating different diseases with the same method".

Humans ; Clavicle ; Fractures, Bone/surgery*

Objective:To investigate the 10-year outcome and prognostic factors of laparo-scopic D 2 radical distal gastrectomy for locally advanced gastric cancer. Methods:The retrospec-tive cohort study was conducted. The clinicopathological data of 652 patients with locally advanced gastric cancer who were admitted to 16 hospitals from the multicenter database of laparoscopic gastric cancer surgery in the Chinese Laparoscopic Gastrointestinal Surgery Study (CLASS) Group, including 214 cases in the First Affiliated Hospital of Army Medical University, 191 cases in Fujian Medical University Union Hospital, 52 cases in Nanfang Hospital of Southern Medical University, 49 cases in West China Hospital of Sichuan University, 43 cases in Xijing Hospital of Air Force Medical University, 25 cases in Jiangsu Province Hospital of Chinese Medicine, 14 cases in the First Medical Center of the Chinese PLA General Hospital, 12 cases in No.989 Hospital of PLA, 12 cases in the Third Affiliated Hospital of Sun Yat-Sen University, 10 cases in the First Affiliated Hospital of Nanchang University, 9 cases in the First People's Hospital of Foshan, 7 cases in Zhujiang Hospital of Southern Medical University, 7 cases in Fujian Medical University Cancer Hospital, 3 cases in Zhongshan Hospital of Fudan University, 2 cases in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2 cases in Peking University Cancer Hospital & Institute, from February 2004 to December 2010 were collected. There were 442 males and 210 females, aged (57±12)years. All patients underwent laparoscopic D 2 radical distal gastrectomy. Observation indicators: (1) surgical situations; (2) postoperative pathological examination; (3) postoperative recovery and complications; (4) follow-up; (5) prognostic factors analysis. Follow-up was conducted by outpatient examination and telephone interview to detect the tumor recurrence and metastasis, postoperative survival of patients up to March 2020. Measurement data with normal distribution were represented as Mean± SD, and measurement data with skewed distribution were represented as M( Q1, Q3) or M(range). Count data were described as absolute numbers or percen-tages, and comparison between groups was conducted using the chi-square test. Comparison of ordinal data was analyzed using the rank sum test. The life table method was used to calculate survival rates and the Kaplan-Meier method was used to draw survival curves. Log-Rank test was used for survival analysis. Univariate and multivariate analyses were analyzed using the COX hazard regression model. Results:(1) Surgical situations: among 652 patients, 617 cases underwent D 2 lymph node dissection and 35 cases underwent D 2+ lymph node dissection. There were 348 cases with Billroth Ⅱ anastomosis, 218 cases with Billroth Ⅰ anastomosis, 25 cases with Roux-en-Y anastomosis and 61 cases with other digestive tract reconstruction methods. Twelve patients had combined visceral resection. There were 569 patients with intraoperative blood transfusion and 83 cases without blood transfusion. The operation time of 652 patients was 187(155,240)minutes and volume of intraoperative blood loss was 100(50,150)mL. (2) Postoperative pathological examina-tion: the maximum diameter of tumor was (4.5±2.0)cm of 652 patients. The number of lymph node dissected of 652 patients was 26(19,35), in which the number of lymph node dissected was >15 of 570 cases and ≤15 of 82 cases. The number of metastatic lymph node was 4(1,9). The proximal tumor margin was (4.8±1.6)cm and the distal tumor margin was (4.5±1.5)cm. Among 652 patients, 255 cases were classified as Borrmann type Ⅰ-Ⅱ, 334 cases were classified as Borrmann type Ⅲ-Ⅳ, and 63 cases had missing Borrmann classification data. The degree of tumor differentiation was high or medium in 171 cases, low or undifferentiated in 430 cases, and the tumor differentiation data was missing in 51 cases. There were 123, 253 and 276 cases in pathological stage T2, T3 and T4a, respectively. There were 116, 131, 214 and 191 cases in pathological stage N0, N1, N2 and N3, respectively. There were 260 and 392 cases in pathological TNM stage Ⅱ and Ⅲ, respectively. (3) Postoperative recovery and complications: the time to postoperative first out-of-bed activities, time to postoperative first flatus, time to the initial liquid food intake, duration of postoperative hospital stay of 652 patients were 3(2,4)days, 4(3,5)days, 5(4,6)days, 10(9,13)days, respectively. Among 652 patients, 69 cases had postoperative complications. Clavien-Dindo grade Ⅰ-Ⅱ, grade Ⅲa, grade Ⅲb, and grade Ⅳa complications occurred in 60, 3, 5 and 1 cases, respectively (some patients could have multiple complications). The duodenal stump leakage was the most common surgical complication, with the incidence of 3.07%(20/652). Respiratory complication was the most common systemic complication, with the incidence of 2.91%(19/652). All the 69 patients were recovered and discharged successfully after treatment. (4) Follow-up: 652 patients were followed up for 110-193 months, with a median follow-up time of 124 months. There were 298 cases with postoperative recurrence and metastasis. Of the 255 patients with the time to postoperative recurrence and metastasis ≤5 years, there were 21 cases with distant metastasis, 69 cases with peritoneal metastasis, 37 cases with local recurrence, 52 cases with multiple recurrence and metastasis, 76 cases with recurrence and metastasis at other locations. The above indicators were 5, 9, 10, 4, 15 of the 43 patients with the time to postoperative recurrence and metastasis >5 years. There was no significant difference in the type of recurrence and metastasis between them ( χ2=5.52, P>0.05). Cases in pathological TNM stage Ⅱ and Ⅲ were 62 and 193 of the patients with the time to postoperative recurrence and metastasis ≤5 years, versus 23 and 20 of the patients with the time to postoperative recurrence and metastasis >5 years, showing a significant difference in pathological TNM staging between them ( χ2=15.36, P<0.05). Cases in pathological stage T2, T3, T4a were 42, 95, 118 of the patients with the time to postoperative recurrence and metastasis ≤5 years, versus 9, 21, 13 of the patients with the time to postoperative recurrence and metastasis >5 years, showing no significant difference in pathological T staging between them ( Z=-1.80, P>0.05). Further analysis showed no significant difference in cases in pathological stage T2 or T3 ( χ2=0.52, 2.08, P>0.05) but a significant difference in cases in pathological stage T4a between them ( χ2=3.84, P<0.05). Cases in pathological stage N0, N1, N2, N3 were 19, 44, 85, 107 of the patients with the time to postoperative recurrence and metastasis ≤5 years, versus 12, 5, 18, 8 of the patients with the time to postoperative recurrence and metastasis >5 years, showing a significant difference in pathological N staging between them ( Z=-3.34, P<0.05). Further analysis showed significant differences in cases in pathological stage N0 and N3 ( χ2=16.52, 8.47, P<0.05) but no significant difference in cases in pathological stage N1 or N2 ( χ2=0.85, 1.18, P>0.05). The median overall survival time was 81 months after surgery and 10-year overall survival rate was 46.1% of 652 patients. The 10-year overall survival rates of patients in TNM stage Ⅱ and Ⅲ were 59.6% and 37.5%, respectively, showing a significant difference between them ( χ2=35.29, P<0.05). In further analysis, the 10-year overall survival rates of patients in pathological TNM stage ⅡA, ⅡB, ⅢA, ⅢB and ⅢC were 65.6%, 55.8%, 46.9%, 37.1% and 24.0%, respectively, showing a significant difference between them ( χ2=55.06, P<0.05). The 10-year overall survival rates of patients in patholo-gical stage T2, T3 and T4a were 55.2%, 46.5% and 41.5%, respectively, showing a significant difference between them ( χ2=8.39, P<0.05). The 10-year overall survival rates of patients in patholo-gical stage N0, N1, N2 and N3 were 63.7%, 56.2%, 48.5% and 26.4%, respectively, showing a signifi-cant difference between them ( χ2=54.89, P<0.05). (5) Prognostic factors analysis: results of univariate analysis showed that age, maximum diameter of tumor, degree of tumor differentiation as low or undifferentiated, pathological TNM staging, pathological T staging, pathological stage N2 or N3, post-operative chemotherapy were related factors for the 10-year overall survival rate of locally advanced gastric cancer patients undergoing laparoscopic D 2 radical distal gastrectomy ( hazard ratio=1.45, 1.64, 1.37, 2.05, 1.30, 1.68, 3.08, 0.56, 95% confidence interval as 1.15-1.84, 1.32-2.03, 1.05-1.77, 1.62-2.59, 1.05-1.61, 1.17-2.42, 2.15-4.41, 0.44-0.70, P<0.05). Results of multivariate analysis showed that maximum diameter of tumor >4 cm, low-differentiated or undifferentiated tumor, pathological TNM stage Ⅲ were independent risk factors for the 10-year overall survival rate of locally advanced gastric cancer patients undergoing laparoscopic D 2 radical distal gastrectomy ( hazard ratio=1.48,1.44, 1.81, 95% confidence interval as 1.19-1.84, 1.11-1.88, 1.42-2.30, P<0.05) and postoperative chemotherapy was a independent protective factor for the 10-year overall survi-val rate of locally advanced gastric cancer patients undergoing laparoscopic D 2 radical distal gastrec-tomy ( hazard ratio=0.57, 95% confidence interval as 045-0.73, P<0.05). Conclusions:Laparoscopic assisted D 2 radical distal gastrectomy for locally advanced gastric cancer has satisfactory 10-year oncologic outcomes. A high proportion of patients in pathological TNM stage Ⅲ, pathological stage T4a, pathological stage N3 have the time to postoperative recurrence and metastasis ≤5 years, whereas a high proportion of patients in pathological TNM stage Ⅱ or pathological stage N0 have the time to postoperative recurrence and metastasis >5 years. Maximum diameter of tumor >4 cm, low-differentiated or undifferentiated tumor, pathological TNM stage Ⅲ are independent risk factors for the 10-year overall survival rate of locally advanced gastric cancer patients undergoing laparos-copic D 2 radical distal gastrectomy. Postoperative chemotherapy is a independent protective factor for the 10-year overall survival rate of locally advanced gastric cancer patients undergoing laparos-copic D 2 radical distal gastrectomy.