Objective To optimize the technical parameters used for embryo transfer in golden hamsters,and explore the optimal number and developmental period of transplanted embryos for golden hamster pregnancy recipients.Methods We established a population of true pregnant albino golden hamster embryo transfer recipients and compared the effects of different embryonic developmental stages,recipient embryo reduction,and secondary transfer recipients on litter yield,donor embryo yield,and offspring survival rate.Results Compared with wild-type golden hamster transplant recipients,true pregnancy albino recipients allowed the origin of the offspring to be determined quickly,and were suitable for various reproductive experimental programs based on embryo transfer.The use of fertilized eggs or two-cell embryos had no significant impact on the transfer effect(P＞0.05);however,the rate of donor embryos was significantly increased in the recipient embryo-reduction group(22％,P＜0.05).The second transfer recipient's non-pregnancy rate was significantly increased(42％,P＜0.01).The highest embryo yield rate(27％,P＜0.01)and normal survival rate(89％)occurred with the transfer of 6～10 embryos.Conclusions The transfer of 6～10 donor embryos may improve the yield and survival rate of donor embryos.Here,we successfully established an embryo transfer method using pregnant albino golden hamsters as recipients,thus providing technical support for the application and development of gene modification models in golden hamsters.

Objective To establish an ideal model of the mitral valve,including the left heart and blood,and study the motion characteristics of the mitral valve in blood flow using the fluid-structure interaction(FSI)simulation.Methods Based on anatomical parameters,models of the mitral valve,left heart,and blood were established.The finite-elements combined immersed boundary method was used for FSI to simulate the motion of the mitral valve using the LS-DYNA software.Morphological,mechanical,and hemodynamic parameters were compared with those obtained from structural simulations.Results The morphological results of the mitral valve from the two simulations differed significantly,and the FSI results matched the ultrasound images.The stress distributions of the leaflets in the FSI and structural simulations were consistent.The maximum first principal stresses calculated by FSI and structural simulations were 1.48 MPa and 1.53 MPa,respectively,with a relative error of 3.27％.The fluid field in the left heart was complex with vortex structures,and the maximum mitral flow velocity was 1.02 m/s during diastole,consistent with the physiological data of healthy humans(0.89±0.15 m/s).Conclusions The morphological results of the mitral valve obtained from the FSI simulation were closer to those in the physiological state.FSI simulations can provide flow patterns that are indispensable for clinical diagnosis.Structural simulations are more efficient for studying leaflet stress distribution.

Objective:To evaluate the efficacy and safety of antaitasvir phosphate 100 mg or 200 mg combined with yiqibuvir for 12 weeks in patients with various genotypes of chronic hepatitis C, without cirrhosis or compensated stage cirrhosis.Methods:Patients with chronic hepatitis C (without cirrhosis or compensated stage cirrhosis) were randomly assigned to the antaitasvir phosphate 100 mg+yiqibuvir 600 mg group (100 mg group) or the antaitasvir phosphate 200 mg+yiqibuvir 600 mg group (200 mg group) in a 1∶1 ratio. The drugs were continuously administered once a day for 12 weeks and observed for 24 weeks after drug withdrawal. The drug safety profile was assessed concurrently with the observation of the sustained virological response (SVR12) in the two patient groups 12 weeks following the drug cessation. The intention-to-treat concept was used to define as closely as possible a full analysis set, including all randomized cases who received the experimental drug at least once. The safety set was collected from all subjects who received the experimental drug at least once (regardless of whether they participated in the randomization group) in this study. All efficacy endpoints and safety profile data were summarized using descriptive statistics. The primary efficacy endpoint was SVR12. The primary analysis was performed on a full analysis set. The frequency and proportion of cases were calculated in the experimental drug group (antaitasvir phosphate capsules combined with yiqibuvir tablets) that achieved "HCV RNA

Objective: To investigate the changes of osteogenic genes and osteogenic capacity after overexpression of bone morphogenetic protein 2(Bmp2) in rat adipose stem cells． Methods: Bmp2 was transfected into rat adipose stem cells by electrotransfection，and the blank group was used as control．The cells were divided into blank group ( Control group) ，empty load group (Vector group) ，osteogenesis induction group (Inducement group) ，and overexpression group (Bmp2 group) ．Western blot，qRT-PCR and immunofluorescence were used to compare the expression of osteogenic genes，and alkaline phosphatase staining and alizarin red staining were used to observe cobalt sulfide precipitation and calcium salt deposition. Results : Western blot and qRT-PCR showed that the expression of Bmp2，Bmp4，OPN，OCN，Runx2，P-Smad1 /5，Dlx2，and BSP was significantly higher compared to the Control group (P ＜0. 05 ) ，while the expression of Smad1 /5 was not statistically different． Immunofluorescence showed that the fluorescence intensity of OPN and Runx2 was significantly higher compared to the Control group (P ＜0. 05) ．The alizarin red staining and alkaline phosphatase staining showed that the Bmp2 group had increased pericellular calcium salt deposition，increased cobalt sulfide precipitation，and enhanced alkaline phosphatase activity compared to the Control group． Conclusion Overexpression of Bmp2 enhances osteogenic gene expression in adipose stem cells and improves their osteogenic ability.

Objective To make biomechanical evaluation on ultimate pullout strength of the suture anchors based on the angle of suture anchor (SA) implanted into the humerus during arthroscopic rotator cuff repair (RCR) surgery. Methods Polyurethane materials with densities of 0.16 g/cm3 and 0.32 g/cm3 were used to simulate osteoporosis and normal cancellous bone, and polyurethane materials with densities of 0.64 g/cm3 and 3 mm thickness were used to simulate human cortical bone. The two kinds of cancellous bone models were respectively adhered together with cortical bone model to construct human humerus model. Titanium metal suture anchors were inserted into humerus models at 45°, 60°, 75° and 90° angle, then the continuous tensile experiments were performed, and 45° pulling direction between the humerus model surface and suture anchor was used to simulate the supraspinatus physiological traction direction, and each group was continuously tested 8 times, recording the pullout strength and failure modes. ResultsThe pullout force of high-density bone models was significantly higher than that of low-density bone models (P＜0.001), and at the same density, compared with 45°, 60° and 75°, the implant angle of 90° has a larger pullout force (P＜0.01). Conclusions In the model of humerus, the 90° implantation of suture anchor showed better biomechanical properties, and the vertical implantation of anchor in the repair of rotator cuff was beneficial to the knotting during operation and postoperative recovery of the supraspinatus.

To monitor genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV),RT-PCR was used to identify a sample suspected of PRRSV infection.A PRRSV named SC-GY strain was obtained,and its Nsp2,ORF5 and ORF3 genes were used for sequence alignment and phylogenetic tree construction.The results showed that SC-GY strain is highly pathogenic PRRSV American variant strains with Nsp2 gene discontinuous deletion of 30 amino acids,ORF3 gene aa17 a serine (S) insert.Comparing to VR2332,CH-1a,JXA1,HUN4,NADC30,HENAN-XINX and SC2012,the Nsp2,ORF5 and ORF3 of SC-GY shared 70.3％-97.9％,82.4％-97.6％ and 83.1％-98.2％ of nucleotide similarity,and 62.3％-96.3％,78.0％-95.7％ and 81.6％-96.5％ of deduced amino acid similarity;and compared to LV they shared only 18.9％,60.8％ and 63.7％ of nucleotide similarity,and 14.0％,54.9％ and 57.2％ of deduced amino acid similarity.The phylogenetic tree revealed that the SC-GY formed independent small branches although it belonged to the same subgroup as highly pathogenic PRRSV strains.The results showed that in high frequency live vaccine immunization of currently PRRSV,the gene of PRRSV epidemic strain is still in constant variation.Vaccination of live PRRSV vaccines should be reduced and surveillance of PRRSV strains should be enhanced.

ObjectiveTo summarize the operative experiences and postoperative complications of pediatric liver transplantation (LT).MethodsClinical data of 88 child patients undergoing LT in West China Hospital, Sichuan University between January 2000 and October 2015 were retrospectively analyzed. Among the patients, 38 were males and 50 were females, with the age ranging from 3 months to 17 years and 7 months old and the median of 2 years and 3 months old. Seventy-seven cases underwent living donor LT and 11 underwent cadaver LT. Thirty-nine cases underwent left lobe transplantation and 49 underwent left lateral lobe transplantation. The main immunosuppressive regimen after operation was adrenocortical hormone + tacrolimus (FK506) or cyclosporin. And heparin was used after operation for anticoagulation. The informed consents of all patients and their families were obtained and the local ethical committee approval was received. The intraoperative conditions and postoperative complications of the patients were observed.ResultsThe average operation time was (7.5±2.4) h, the median intraoperative blood loss was 475(100-2 000) ml, the transfusion of red blood cells was 1.8(0-6.5) U, the warm ischemia time was 2.5(1.0-10.0) min, the cold ischemia time was 188(110-590) min and the inferior vena cava occlusion time was 75(35-170) min. Forty-nine cases developed early complications after operation, and the incidence of vascular complications was 11%(10/88), including 4 cases of hepatic artery thrombosis, 5 of portal vein thrombosis and 1 of hepatic vein stenosis. Sixteen cases developed late complications, and the incidence of biliary complications was 12% (11/88), including 9 cases of cholangitis, 1 of bile leakage and 1 of bile duct stricture. The 1-, 3-year survival rate after LT was respectively 81% and 75%.Conclusions The success rate and postoperative survival rate of pediatric LT have increased signiifcantly in recent years. However, the incidence of postoperative vascular and biliary complications are still high. Thus, the operation procedures should be kept improving, and the immunosuppressant and anticoagulant therapy should be individually adjusted in order to obtain better efifcacy.

BACKGROUNDTobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most important carcinogen in cigarette. Models induced by NNK are widely used in investigations about the mechanisms of pulmonary neoplasia and chemoprevention studies. The aim of this study is to explore the pulmonary precancerous lesions induced by NNK and its possible mechanisms.

METHODSFifteen Wistar rats were divided into two trial groups, in which the high-dose group was instilled with iodized oil including 10 mg (50 mg/kg) NNK into the left lower lobar bronchus, and the low-dose group received 5mg ( 25mg/kg) NNK. Another 15 Wistar rats were instilled only with iodized oil as control group. All rats were examined immediately after instillation and followed up periodically by pulmogram. The pulmonary tissues of rats were pathologically examined, and the expression of AE1/AE3, PCNA and p53 was detected by immunohistochemical method.

RESULTSThe pulmograms showed that the iodized oil localized at the bottom of left lobe and disappeared 107 days later. In trial group, 10 of 15 rats (67%) had nodus at the bottom of left lobe. All of rats in trial group (15/15) displayed atypical hyperplasia in alveolar region, showing single or multiple layers of proliferative epithelial cells along intact alveolar septa with irregular and non-discrete margins of lesion, but continuous alveolar spaces were not obliterated by proliferative epithelial cells. Ten of 15 rats in trial group showed severe atypical hyperplasia of glandular epithelium with occasional infiltrating to muscular layer. All of those atypical hyperplasia cells showed positive AE1/AE3 expression. The positive rate of PCNA was 90% (9/10) and 100% (5/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (13%, 2/15) (P=0.000, P=0.001). The positive rate of p53 expression was 50% (5/10) and 60% (3/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (0) (P=0.005, P=0.009). However, there was no remarkable difference in PCNA and p53 expression between low-dose group and high-dose group (P > 0.05).

CONCLUSIONSTransbronchial instillation of iodized oil including tobacco-specific NNK can induce pulmonary lesions as atypical hyperplasia of alveolar cell and glandular epithelium in Wistar rats. This model can be used in experimental studies about tobacco-related lung cancer.

BACKGROUNDTo study the inhibition effects of both extraneous right sense and antisense p53 on malignant phenotype of human lung cancer cell line.

METHODSThe named 801D cell line with p53 deletion and mutation at 248 code was selected as a model in vitro. The recombined plasmid pEGFP-p53(RS) and pEGFP-p53(AS) were constructed. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohistochemical stain of p53 antibody. The inhibition effect of extraneous p53 on tumor growth in vitro were determined by clonogenic survival assay. FCM analysis was carried out in cells. The inhibition effect on malignant growth of extraneous p53 in vivo was observed by heteroplastic transplant on nude mouse.

RESULTSThe transfected cell lines, pEGFP-p53(AS)-801D, pEGFP-p53(RS)-801D and pEGFP-801D were established. Presence of extraneous p53 and neo genes in pEGFP-p53(AS)-801D and pEGFP-p53(RS)-801D was proved by PCR and green fluorescence was found out in those cells under the microscope. Mutant protein in pEGFP-p53(AS)-801D was negative by immunohistochemical stain. The malignant growth of these transfected cell lines was inhibited comparing with parents in vivo and in vitro. Inhibition rate of colony formation was 62.0% for pEGFP-p53(AS)-801D and 80.8% for pEGFP-p53(RS)-801D. The tumorigenicity in nude mice was suppressed. Inhibition effects of extraneous right sense p53 on malignant growth of 801D was more distinct. FCM analysis showed that pEGFP-p53(AS)-801D cells were arrested at G1 phase.

CONCLUSIONSThe transfected cell lines with extraneous right sense and antisense p53 appear that malignant growth can be inhibited in vivo and in vitro.

BACKGROUNDTo construct and express a phage display library of anti human lung cancer monoclonal antibody 5F-11.

METHODSImmunoglobulin variable regions (VH,VL) were amplified from 5F-11 hybridrom by RT-PCR. ScFv genes consisting of VH DNA and VL DNA joined together by a linker DNA were cloned into a phage vector pCANTAB5E. After 4 rounds of screening with lung adenocarcinoma cell line A2 as antigen, an enriched secondary phage display library was obtained.

RESULTSA recombinant phage display library with total of 8×10⁷ pfu/ml was established. Randomized clones from unselected library digested with BstNⅠ showed different patterns, however, those from selected library showed that phages with special pattern were enriched. Twenty-three out of 30 clones were found to respond strongly to A2 cell lines.

CONCLUSIONSThe ScFv of anti-lung adenocarcinoma monoclonal antibody 5F-11 can be successfully produced, which may be useful to widen the application of the antibody.