Objective To investigate the clinical application of multiplex allele-specific PCR assays for simultaneous detection of the mitochondrial 12S rRNA A1555G and C1494T mutations associated with aminoglycoside-induced hearing impairment.Methods Three standard plasmids of different genotypes (wild-type, A1555G mutant and C1494T mutant) were constructed for templates and allele-specific primers aiming directly at wild-type and mutant of mitochondrial DNA nt1555 and nt1494 were designed for developing a multiplex allele-specific PCR technique to detect the A1555G and C1494T mutations.Then the method was applied to clinical screening of 138 non-syndromic hearing loss subjects and confirmed by DNA sequencing.Results Multiplex allele-specific PCR was successfully applied to the detection of A1555G and C1494T mutations in a cohort of 138 Han Chinese genetically unrelated hearing-loss subjects.Finally, 11(7.97%) unrelated affected subjects harbored the A1555G and C1494T mutations in the 12S rRNA gene(10 cases for A1555G and 1 cases for C1494T), which was well consistent with results of DNA sequencing [7.97%(11/138), Kappa=1.000, P＜0.01].Conclusion This study indicates that the multiplex allele-specific PCR assay is useful, convenient and reliable in the detection of the A1555G and C1494T mutations, which could identify the subjects at risk and effectively prevent of aminoglycoside-induced hearing loss.

OBJECTIVETo investigate the anti-tumor activity of CCL21-exCD40L eukaryotic expression vector.

METHODSCCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4, and then was cloned into expression vector pcDNA3.1(+). pcDNA3.1(+)/CCL21 and pcDNA3.1(+)/exCD were constructed as negative control. Wsestern blot was used to identify the fusion protein. CHO cells was transfected with pcDNA3.1(+)/CCL21-exCD, pcDNA3.1(+)/CCL21 and pcDNA3.1(+), respectively. The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.

RESULTSGene sequencing and restrictive digestion proved the successful construction of pcDNA3.1(+)/CCL21-exCD40L,and its expression was conformed by western blot. The transfectant supernantes of pcDNA3.1(+)/CCL21-exCD40 group had a significant chmotactic function to DCs, of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field. After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse, the tumor mass reduced remarkablely, and all the mouse in fusion gene group survived after 4 weeks.

CONCLUSIONCCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all control group.

Animals ; CD40 Ligand ; genetics ; pharmacology ; CHO Cells ; Cell Line, Tumor ; Chemokine CCL21 ; genetics ; pharmacology ; Colonic Neoplasms ; therapy ; Cricetulus ; Dendritic Cells ; drug effects ; physiology ; Genetic Therapy ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology

OBJECTIVETo construct the murine CCL21 eukaryotic expression plasmid, and to investigate the chemotactic function of its products.

METHODSMurine CCL21 cDNA was amplified by RT-PCR from murine total RNA, and was inserted into eukaryotic expression plasmid pcDNA3.1 after confirmation of sequencing. The recombinant CCL21 plasmid was transferred into mouse forestomach carcinoma (MFC) cells and the chemotactic function of expressed products was detected by chemotaxis assay.

RESULTGene sequencing, gel electrophoresis of PCR products and restrictive digestion proved the successful construction of CCL21, and its expression was confirmed by Western Blot. The transfected tumor cells had a significant chemotactic function to DC.

CONCLUSIONThe recombinant murine CCL21 eukaryotic expression plasmid has been successfully constructed, and its expression products in tumor cells have a marked chemotactic function to DC.

Animals ; Base Sequence ; Chemokine CCL21 ; biosynthesis ; genetics ; Chemotaxis, Leukocyte ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendritic Cells ; drug effects ; immunology ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Stomach Neoplasms ; metabolism ; pathology ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo evaluate the effect and complication of surgical treatment for Pilon fracture using the posterolateral approach.

METHODSFrom August 2009 to March 2011, 15 patients with Pilon fractures (2 in B3,13 in C) and with a separate displaced posterior malleolar fragment was treated in two-stage: the first stage management was on stabiliztion of the soft tissue envelope with temporary external fixator of spanning arthritis, and the second stage management was open reduction and internal fixation with posterolateral approach and anteromedial or anteralateral approach.

RESULTSAll patients were followed-up for 12 to 17 months (14.2 months in average). Thirteen of the 15 fractures healed, but 2 fractures needed autologous bone graft procedure duo to nonuion. There was no wound complication related to poterolateral incion. Fourteen fractures had less than 2 mm of incongruity of distal tibia joint. According to Baired-Jackson criteria, the results were excellent in 2 cases, good in 7, fair in 4, and poor in 2.

CONCLUSIONThe posterolateral approach offers direct visualization for the reduction and fixation of the fibula and posterior distal fragment of the tibia Pilon fractures, faciliate the management of this difficult fracture pattern.

Adult ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Tibial Fractures ; surgery

Adult ; Aged ; Aged, 80 and over ; Decompression, Surgical ; Female ; Fractures, Bone ; complications ; surgery ; Humans ; Leg Injuries ; complications ; surgery ; Male ; Middle Aged

The promotion or inhibition of blood vessels by TCM, active ingredients of TCM, TCM compounds, and Chinese patent drugs is currently a research hotspot, with relatively abundant research. However, the study of the role and profound mechanism of TCM on angiogenesis after cerebral ischemia is relatively rare. Therefore, this article reviewed theories, in vitro studies and in vivo studies on the promotion of cerebral angiogenesis by TCM, and summarized the studies on TCM regulation of angiogenesis through Notch and Wnt signaling pathway mechanisms, with a purpose to provide references for resarch on related mechanisms and development of new deugs.

OBJECTIVESTo discuss the diagnosis and differential diagnosis, and to establish an effective protocol to treat the posterior Monteggia fracture-dislocations of proximal ulna in adult according to our experience.

METHODSBetween April 2004 and December 2007, 16 patients with posterior Monteggia fracture-dislocations were treated surgically, 13 were followed up at a mean of 28 months (range, 12 - 58 months). All the operations were through the posterior midline approach. The fractures of radial head and coronoid process were reduced and fixed, if possible. The proximal ulna fractures were fixed with a single plate in 7 cases, plate combined with K-wires in 2, plate combined with K-wires tension band in 3, and K-wires tension band combined with screws in 1.

RESULTSNo elbow was painful or unstable at the last follow up examination. They had an average of 100 degrees (range, 0 degrees to 145 degrees ) of flexion-extension of elbow. The average motion of forearm rotation was 119 degrees (range, 0 degrees to 170 degrees ). The mean Mayo Elbow Performance Score (MEPS) was 93.1 points (67 - 100 points), excellent and good results were achieved in 92.3%. The mean system of Broberg and Morrey score was 88.8 points (53 - 100 points), excellent and good results were achieved in 76.9%.

CONCLUSIONSAttention should be paid to the diagnosis and differential diagnosis of the posterior Monteggia fracture-dislocation of proximal ulna. Anatomically reduction and stable fixation of proximal ulna is the keystone for the surgical treatment.

Adult ; Bone Plates ; Bone Screws ; Bone Wires ; Diagnosis, Differential ; Elbow Joint ; surgery ; Follow-Up Studies ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Monteggia's Fracture ; diagnosis ; surgery ; Treatment Outcome

OBJECTIVETo evaluate the expression of TEIF protein in human tumors of soft tissue and its significance.

METHODSAnti-TEIF polyclonal antibody was generated by immunization of E.coli expressed His-TEIF protein. The expression of TEIF in 166 cases of sarcomas and 28 case benign tumors or tumor-like lesions of soft tissue arranged in tissue chip was analyzed by immunohistochemistry.

RESULTSPolyclonal antibody obtained from immunized rabbit was verified in Western blot to prove its specific reactivity with native TEIF protein. The immunohistochemical staining of TEIF showed that about 58% (97/166) of sarcomas were positive and significantly different from that of benign tumors or tumor-like lesions (11%, 3/28). The positive staining was predominantly in synovial sarcoma 94% (16/17), primitive neuroectodermal tumor (PNET) 91% (21/23), both of which were significantly higher than 43% (6/14) of dermatofibrosarcoma protuberans, 38% (6/16) of myxofibrosarcoma, 36% (8/22) of malignant peripheral nerve sheath tumor, 32% (6/19) of liposarcoma, (P < 0.05, respectively), but not higher than 75% (15/20) of malignant fibrous histiocytoma, 70% (7/10) of rhabdomyosarcoma or 64% (9/14) of leiomyosarcoma. Meanwhile, strong positive staining of TEIF (>or= 2+) was frequently observed in PNET (83%, 19/23) and synovial sarcoma (76%, 13/17). With respect to FNCLCC grading, 19 cases of grade I sarcoma TEIF was 32% (6/19) and strong positive was 11% (2/19), 44 cases of grade II sarcoma was 48% (21/44) and 32% (14/44), and 70 of grade III was 84% (59/70) and 70% (49/70). The rate of either positive or strong positive in grade III sarcoma was significantly different from that of either grade I or II (P < 0.05), but no difference between the latter two groups (P > 0.05).

CONCLUSIONSTEIF protein could be detected in large part of soft tissue sarcomas, and it not only over-expressed in most of PNET, synovial sarcomas, but also correlated with histological grading.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Blotting, Western ; Cell Line, Tumor ; Child ; Child, Preschool ; Female ; HeLa Cells ; Histiocytoma, Malignant Fibrous ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Infant ; Leiomyoma ; metabolism ; pathology ; Male ; Middle Aged ; Neuroectodermal Tumors, Primitive ; metabolism ; pathology ; Rhabdomyosarcoma ; metabolism ; pathology ; Sarcoma ; metabolism ; pathology ; Sarcoma, Synovial ; metabolism ; pathology ; Soft Tissue Neoplasms ; metabolism ; pathology ; Tissue Array Analysis ; Transcription Factors ; biosynthesis ; Young Adult

OBJECTIVETo compare the expressions of lysyl oxidase (LOX) and matrix metalloproteinases-2 (MMP-2) in gastric cancer and pericancerous tissues, in gastric cancers with and without lymph node metastasis, and to analyze the effects of LOX and MMP-2 on tumor invasion and metastasis.

METHODSGastric cancer and pericancerous tissues were collected from 46 patients who underwent surgery. Levels of LOX and MMP-2 mRNA were detected by RT-PCR. Protein abundance of LOX and MMP-2 was examined using Western blot.

RESULTSExpressions of LOX and MMP-2 mRNA, and protein in 46 gastric cancers were significantly higher than that in 46 pericancerous tissues. In gastric cancer with lymph node metastasis, the levels of LOX and MMP-2 mRNA and protein were higher than those in gastric cancers without lymph node metastasis (P < 0.05). In the groups of gastric cancer with lymph node metastasis, expression of LOX was positively correlated with MMP-2 protein expression (P < 0.01).

CONCLUSIONSExpressions of LOX and MMP-2 in gastric cancer tissues are significantly higher than that in pericancerous tissues. The expressions of LOX and MMP-2 in gastric cancer with lymph node metastasis are higher than that in gastric cancer without lymph node metastasis. Expressions of LOX and MMP-2 are positively correlated. The results suggest that LOX and MMP-2 may promote the growth and metastasis of gastric cancer.

Adenocarcinoma ; metabolism ; pathology ; surgery ; Biomarkers, Tumor ; metabolism ; Female ; Gastrectomy ; Humans ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Middle Aged ; Neoplasm Invasiveness ; Protein-Lysine 6-Oxidase ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stomach ; metabolism ; surgery ; Stomach Neoplasms ; metabolism ; pathology ; surgery

OBJECTIVE Paeoniflorin (PF) and albiflorin (AF) are the major active components of total peony glucosides(TPG)from Paeonia lactiflora Pal,which have many biological activities such as anti-inflammatory, antioxidation and anti-hypertension effects. The drug-drug pharmacokinetic interaction among PF,AF and TPG,the pharmacokinetic comparisons of AF between hypoxia and normoxia,the transport of AF cross the blood-brain barrier cell model and the transport of AF/PF/TPG cross Caco-2 cell model were investigated.METHODS A highly sensitive and rapid UPLC-MS method with multiple-reaction monitoring(MRM)scanning via electrospray ionization(ESI)source operating both in the positive and negative ionization mode was successfully developed and validated for simultaneous quantitation of PF and AF in rat plasma after an oral administration of PF,AF and TPG. RESULTS The validated and developed UPLC-MS/MS method was successfully applied to simultaneously determine the AF and PF concentration in rat plasma and investigate pharmacokinetic interactions after a single intragastrical ad-ministration of PF,AF,co-administration of PF with AF and TPG,respectively.The elimination of both PF co-administered with AF and PF in TPG were slower than those for PF alone and the distribution in the tissues was wider.The combination of PF with AF or TPG could significantly increase the values of the AUC, MRT and t1/2of the drug PF, and reduce the values of CL of PF. From a comparison of the main pharmacokinetic parameters among AF alone, AF combined with PF and AF in TPG, the values of the MRT and t1/2of AF in TPG were greater than that of AF alone,and there were statistically signifi-cant differences in these parameters(P<0.05,P<0.01).It was also noticed that AUC and Cmaxof PF in hypoxia rats were significantly decreased compared with that of normaxia rats, suggesting that there was a decreased exposure of PF in rats under hypoxia. The multiple active components in TPG may lead to DDIs between some P-gp substrates. CONCLUSION The clinical performance of total peony glucosides would be better than that of single constitute. The outcomes of the study are expected to serve as a basis for development of clinical guidelines on total peony glucosides usage.