ObjectiveTo investigate the effects of Danhong injection on mitochondrial dynamics， morphology， and function in the rat model of chronic heart failure by mediating the adenosine 5'-monophosphate （AMP）-activated protein kinase （AMPK）/dynamin-related protein 1 （Drp1） pathway. MethodsFrom 75 SD rats， 15 rats were randomly selected as the sham group， and the remaining 60 rats were used to prepare a rat model of chronic heart failure by abdominal aortic constriction （AAC）. The modeled rats were randomly allocated into model， Danhong Injection （6 mL·kg^-1）， and captopril （8.8 mg·kg^-1） groups and administrated with corresponding agents for 15 consecutive days. The levels of N-terminal pro-brain natriuretic peptide （NT-pro BNP）， adenosine diphosphate （ADP）， adenosine triphosphate （ATP）， interleukin （IL）-6， IL-1β， and tumor necrosis factor （TNF）-α and the activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ were determined by enzyme-linked immunosorbent assay. The changes in cardiac function were detected by echocardiography. The ultrastructural changes of myocardial mitochondria were observed by transmission electron microscopy. Western blot was employed to assess the protein levels of AMPK， p-AMPK， Drp1， p-Drp1， optic atrophy 1 （Opa1）， mitofusin （Mfn2）， and fission l （Fis1） in the myocardial tissue. Real-time PCR was performed to determine the mRNA levels of Opa1， Mfn2， and Fis1， and immunohistochemistry to detect the expression of p-AMPK. ResultsCompared with the sham group， the model group showed elevated levels of NT-pro BNP， ADP， TNF-α， IL-6， and IL-1β （P<0.01）， declined ATP level （P<0.01）， weakened activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ （P<0.01）， decreased left ventricular ejection fraction （LVEF） and left ventricular fraction shortening （LVFS） （P<0.01）， and increased left ventricular internal diameter at end-diastole （LVDd） and leaf ventricular internal diameter at end-systole （LVIDs） （P<0.01）. Electron microscopy results showed that the model group presented heavily abnormal myocardial structure， with large areas of myofilament structure destroyed and dissolved， significantly enlarged residual structural gaps， and fragmented mitochondria. Western blot results showed that the model group demonstrated down-regulated protein levels of p-AMPK， Mfn2， and Opa1 （P<0.01） and up-regulated protein levels of p-Drp1 and Fis1 （P<0.01） in the myocardial tissue. Real-time PCR results showed that the model group presented up-regulated mRNA level of Fis1 （P<0.01） and down-regulated mRNA levels of Mfn2 and Opa1 （P<0.01）. Immunohistochemistry results showed reduced expression of p-AMPK in the model group compared with sham group （P<0.01）. Compared with the model group， Danhong injection lowered the levels of NT-pro BNP， ADP， TNF-α， IL-6， and IL-1β （P<0.01）， raised the level of ATP （P<0.01）， increased the activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ （P<0.05， P<0.01）， increased the LVEF and LVFS （P<0.01）， decreased the LVDd and LVIDs （P<0.05， P<0.01）， alleviated mitochondrial damage， up-regulated the protein levels of p-AMPK， Mfn2， and Opa1 （P<0.05， P<0.01）， down-regulated the protein levels of p-Drp1 and Fis1 （P<0.01）， reduced the mRNA level of Fis1 （P<0.01）， elevated the mRNA levels of Mfn2 and Opa1 （P<0.05， P<0.01）， and promoted the expression of p-AMPK （P<0.05）. ConclusionDanhong injection repairs the imbalance of mitochondrial dynamics， restores the mitochondrial function， improves the myocardial energy metabolism， and reduces the inflammatory response by regulating the AMPK/Drp1 pathway， thus improving the cardiac function.

Objective To prepare and identify the monoclonal antibodies against respiratory syn-cytial virus nucleoprotein(RSV N protein). Methods A prokaryotic expression system was used to express recombinant RSV N protein in Escherichia coli (E.coli). BALB/c mice were immunized with the recombi-nant N protein after purification. Monoclonal antibodies (McAbs) against the N protein were sorted from these BALB/c mice and then were further characterized by Western blot, ELISA and immunofluorescence. Furthermore,this study used orthogonal experiment to identify McAbs pairs,which could be used for diagno-sis. Results This study succeeded in obtaining 24 hybridoma cells that stably secreted monoclonal antibod-ies against RSV N protein. These antibodies showed good reactivity in ELISA,of which eight had strong spe-cificity in Western blot and 13 could be used in immunofluorescence. This study obtained two McAbs pairs (12F2/11H8-HRP and 12F2/15A8-HRP) that could be used in RSV detection. Conclusion This study succeeded in screening and preparation of McAbs against RSV N protein and obtaining two potential McAbs pairs for rapid detection of RSV.

Objective To explore the anatomic basis for and clinical outcomes of the internal fixation which preserves the pronator quadratus (PQ) for distal radius fractures.Methods Twenty cadaveric specimens of adult upper extremity were used for this study (14 males and 6 females).The radial and ulnar lengths of PQ,the distal and proximal widths of PQ,the distances from the distal end of PQ to the articular surface of the distal radius and to the transverse line of the wrist,and the width of the bony tunnel of PQ were dissected and measured to study the anatomical features of PQ.A retrospective study was conducted of the 18 distal radius fractures which had been treated from March 2015 to March 2017 by internal fixation with T-shaped anatomic locking compression plate (LCP) with PQ preserved.They were 8 males and 10 females,with an average age of 52.7 years (range,from 28 to 65 years).According to the AO classification,there were 8 cases of type 23-A,5 ones of type 23-B and 5 ones of type 23-C1.The functional outcomes of the wrist were assessed using the Cooney scoring system at the last follow-ups.Results The PQ muscle was flat and like a right angle trapezoid with rich blood vessels.The radial and ulnar lengths of PQ were about 4.60 cm and 4.46 cm;the distal and proximal widths of PQ were about 4.41 cm and 4.48 cm;the distance from the distal end of PQ to the transverse line of the wrist was about 3.61 cm;the widths of the distal and proximal bony tunnels were about 3.08 cm and 1.91 cm.The 18 patients were followed up for 6 to 36 months (average,11.5 months).Bone union was achieved in all the patients after a mean time of 2.5 months (range,from 2 to 3 months).The mean Cooney score for the wrist function was 97.7 (range,from 95 to 100) at the last follow-up,yielding an excellent rate of 100％.Conclusions The transverse line of the distal radius fracture is located between 1/4 and 1/2 of the distal PQ.The bony tunnel of PQ is wide enough.It is feasible to preserve the distal PQ muscle in the internal fixation of distal radius fractures of types 23-A,23-B and 23-C1,because it may lead to rapid recovery of the patients and satisfactory wrist function.

BACKGROUND:Fibroblast-like synoviocytes (FLS) in the synovial lining layer are related to the cell proliferation, invasion, migration and apoptosis as well as bone resorption in rheumatoid arthritis. OBJECTIVE:To compare the migration and invasion abilities of FLS (MH7A) in rheumatoid arthritis and normal FLS (HFLS). METHODS:The capacities of cell migration and invasion were evaluated by Transwell cell migration and invasion assays. The primers of the indicated microRNAs were designed and synthesized, and the expression levels of miRNAs were determined by real-time PCR according to the SYBR?PrimeScript?miRNA RT-PCR Kit instruction. RESULTS AND CONCLUSION:MH7A possessed stronger migration and invasion abilities than HFLS. Compared with HFLS, obviously upregulated miR-132, -155, -203, -223 and -124, and significantly downregulated miR-15a, -16, 18a, -19a, -26a and -146a were found in MH7A. These findings suggest that the differentially expressed 11 kinds of rheumatoid arthritis-associated miRNAs participate in the pathogenesis of rheumatoid arthritis probably by enhancing the migration and invasion capacities of MH7A.

Objective To investigate the clinicopathologic characteristics and prognosis of medullary breast carcinoma.Methods We conducted a retrospective analysis on clinical and pathologic data of 166 patients with medullary breast cancer.Results All the patients were female with a median age of 52 years old.The proportion of patients with stage Ⅰ,Ⅱ and Ⅲ disease was 16.9％,68.1％,15.0％,respectively.The Luminal,HER-2 overexpressing and triple-negative subtypes constituted 31.3％,8.4％,and 60.3％,respectively.There was significant difference in regional lymph node status of medullary breast cancer patients with different molecular types (x2 =18.248,P =0.003),but not in tumor size,TNM stage,histological grade,and expression of Ki67 (all P ＞ 0.05).Multivariate survival analysis indicated that TNM stage was an independent predictor in the prognosis of medullary breast cancer (HR =5.664,P =0.001).All the patients were followed up from 15 months to 145 months with a median follow-up time of 108 months.The 5-year survival rate was 91.5％ and the 10-year survival rate was 87.2％.Conclusions The prognosis of medullary breast cancer is favorable.Personalized treatment according to the TNM stage and histopathologic characteristics achieve a favorable prognosis.

Objective: To investigate the feasibility of molecular diagnostic techniques to guide individualized treatment of lung cancer. Methods:A total of 360 patients with lung cancer confirmed by pathological diagnosis received clinical chemotherapy from Jan 2011 to Dec 2013 were selected as the experimental group who chose chemotherapy drugs for drug?sensitive individualized treat?ment based on molecular diagnostic technology test results. Another 180 patients with lung cancer were selected as the control group who did not carry out individual testing only chose conventional chemotherapy. The efficacy and side effects of drugs were assessed. The progression?free survival and median survival time were followed and recorded, and the survival curve was drawn by the Kaplan?Meier method. Results: There was significant difference in the efficacy between the two groups ( P<0?05) , and there was no signifi?cant difference in side effects of drugs. The progression?free survival time and median survival time were significantly different ( P<0?01) . Conclusions: Molecular diagnostic techniques to guide individual treatment of lung cancer have some clinical significance, it will help to improve the efficacy and worthy of further study.

Objective To study the risk factors related to recurrence of gastrointestinal stromal tumor (GIST) after discontinuing postoperative adjuvant imatinib mesylate (IM) treatment.Methods We retrospectively analyzed our clinical database of 138 GIST patients who received radical resection and subsequent IM adjuvant treatment at the Renji Hospital,Shanghai Jiaotong University School of Medicine between January 2006 and January 2014.Results For the entire Multivariate analysis study group,the overall 5-year recurrent free survival (RFS) rate was 54.5％.There were two tumor characteristics which were independent prognostic factors of GIST treated by postoperative IM:Ki67 index (P =0.005),and serosal invasion (P =0.026).The accuracy of comprehensive evaluation based on the two weighted variables was better than NIH staging criteria(AUC:0.714 vs.0.631).Furthermore,two risk groups were created according to the risk model with 5-year RFS of 81.3％ and 31.1％ as low-risk and high-risk groups,respectively (P ＜0.05).Conclusions For patients with intermediate or high risk in NIH classification,if there was tumor serosal invasion,or if there was no local invasion but Ki67 index ＞ 8％,extended continuous IM adjuvant treatment should be recommended after the primary tumor was radically resected.

The lack of effective in vitro infection model for hepatitis E virus (HEV) has greatly hindered the quantitative analysis of neutralizing titers of anti-HEV antibodies and human sera, thus impeding further studies of HEV-stimulated antibody responses and the immunological mechanisms. In order to improve this situation, the infection of HepG2 cells that are inefficient for HEV replication was continuously monitored until the viral load reached the limit of detection on day 13, the results of which confirmed the feasibility of using this cell line to establish the infection model. Then, neutralization assays of five anti-HEV murine monoclonal antibodies and serum samples collected from four HEV vaccine recipients (collected before and after vaccination) were performed by 96 multi-channel parallel infections, nucleic acid extraction, and qPCR. The results showed that the cell model can be applied for quantitative evaluation of the neutralizing capacity of different antibodies and antiserum samples from HEV vaccine recipients. In this study, we have successfully established a high-throughput in vitro HEV replication model, which will prove to be useful for the evaluation of HEV vaccines and studies of HEV epitopes.
Animals ; Antibodies, Viral ; analysis ; immunology ; Hepatitis Antibodies ; analysis ; immunology ; Hepatitis E ; immunology ; virology ; Hepatitis E virus ; chemistry ; immunology ; physiology ; High-Throughput Screening Assays ; methods ; Humans ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; methods ; Virus Replication

BACKGROUND:Studies have shown that microRNAs (miRNAs) have moderating effect on the renewal and differentiation of cancer stem cels. However, there is no complete understanding on the effect of microRNA-17-92 gene on gastric cancer stem cel renewal and proliferation.
OBJECTIVE:To explore the effect of miRNA-17-92 in promoting self-renewal and proliferation of gastric cancer stem cels.
METHODS:(1) The gradualy reduced miRNAs during gastric cancer stem cel self-renewal were investigated using miRNA array based on RNAs from differentiated and adherent cels. (2) The miRNA-17-92 was constructed and transfected to gastric cancer stem cels. (3) The effects of miRNA-17-92 on the self-renewal of gastric cancer stem cels were studied by tumor sphere assayin vitro. (4) The effects of miRNA-17-92 on the proliferation of gastric cancer stem cels were investigated by MTT assay and colony formation assay.
RESULTS AND CONCLUSION:(1) miR-19b/20a/92a expression gradualy reduced in the adhesion and differentiation of gastric cancer stem cels. (2) The expression of lentivirus carrying miRNA-17-19 gene in MKN28 cels and CD44-/EpCAM- cels were significantly increased; transient transfection of pre-miR-19b/20a/92a increased the expression of CD44-/EpCAM- and MKN28 miRNA, transient transfection of pre-miR-19b/20a/92a antagonists reduced the expression of SGC7901 and CD44+/EpCAM+ miRNA; overexpression of lenti-miR-19b/20a/92a significantly increased the ability of gastric cels to form tumor spheres; chemotherapy drugs prolonged the survival time of lenti-miR-19b/20a/92a-infected cels; transient transfection of pre-miR-19b/20a/92a significantly increased the number of CD44+/EpCAM+ cels, but transfection of pre-miR-19b/20a/92a antagonist reduced the number of CD44+/EpCAM+ cels. (3) MTT proliferation assay showed that gastric cancer cel proliferation rate in miR-19b/20a/92a stably expressing group was faster than that in the control group. Transient transfection of miR-19b/20a/92a precursor accelerated the growth rate of gastric cancer cels, and transient transfection of its antagonist slowed down the growth rate of gastric cancer cels. Colony formation assay showed that transient transfection of miR-19b/20a/92a precursor significantly increased the colony formation number as compared with the control group; transient transfection of miR-19b/20a/92a antagonist reduced the colony formation as compared with the control group. These findings indicate that miR-19b/20a/92a gene presents with continuous deletion in gastric cancer stem cel differentiation process, and miRNA-17-92 gene can promote the renewal and proliferation of gastric cancer stem cels.

BACKGROUND:Adipose-derived mesenchymal stem cells have the ability to self-renew and have pluripotent potential under specific conditions in vitro, which have broad application prospects in clinical practice. However, isolation and culture of adipose-derived mesenchymal stem cells stil appear to have many difficulties and shortcomings. OBJECTIVE:To isolate and culture rabbit adipose-derived mesenchymal stem cells in vitro in order to study their morphology, cellsurface markers and biological properties as wel as to investigate the osteogenic and adipogenic potentials of adipose-derived mesenchymal stem cells in vitro. METHODS:Primary adipose-derived mesenchymal stem cells were isolated from the subcutaneous adipose tissue of posterior cervical region from New Zealand white rabbits and digested by 0.1%col agenase I. The cells were passaged and amplified by the trypsin digestion. The passage 4 adipose-derived mesenchymal stem cells were induced to differentiate after exposure to adipogenic or osteogenic medium. The oil red O staining, alkaline phosphatase and alizarin red staining were used to detect the results. The cellviability was detected by the cellcounting kit 8 method to drawn the growth curve. cellsurface markers were examined using flow cytometry. RESULTS AND CONCLUSION:The adipose-derived mesenchymal stem cells isolated from the subcutaneous adipose tissue of rabbits exhibited a fusiform adherent growth in a vortex pattern, and had a strong capability of proliferation that could be passaged stably to the 10th generation. Flow cytometry results showed that the cells highly expressed CD29, CD90, CD44, but lowly expressed CD45 and CD34. After adipogenic induction, the adipose-derived mesenchymal stem cells were positive for oil red O staining;after osteogenic induction, the cells were both positive for alkaline phosphatase and alizarin red staining. These findings suggest that the adipose-derived mesenchymal stem cells were successful y isolated and cultured from the subcutaneous adipose tissue of rabbits, and these cells are pluripotent with the potential to differentiate into adipocytes and osteoblasts, which are expected to be ideal seed cells for bone tissue engineering.