OBJECTIVESTo construct the expression vector pGenesil-3-shRNA that can express the short hairpin RNAs (shRNA) silencing Kir6.2 gene and to study its influence on the proliferation and invasion of HepG2 cells.

METHODSTwo shRNA silencing Kir6.2 genes were transcript synthesized intracellularly by expressed templates of plasmid vector pSilence-3, and the target sequence of Kir6.2 gene was inserted into the upstream of the reporter gene in order to construct the recombinant plasmid vector pGenesil-3. Plasmids containing 2 different sequences of human Kir6.2 mRNA coding region were constructed and transfected into HepG2 cells by using lipofectamine 2000 methods. The experiment was divided into 4 groups: SK (normal), SK-HK (negative control), SK-K1 (transfected with the interfering sequence 1) and SK-K2 (transfected with the interfering sequence 2) groups. A selected single clone was cultured after screening by G418. The expression of Kir6.2 protein was detected by Western blot. MTT assay and Transwell system were used to observe the proliferation and invasion of HepG2 cells.

RESULTSThe recombinant expression plasmid pGenesil-3 was successfully constructed and underexpression of Kir6.2 gene in HepG2 cells was detected by Western blot. Underexpression of Kir6.2 gene significantly decreased the proliferation and invasion of the HepG2 cells.

CONCLUSIONshRNA can inhibit the expression of Kir6.2 gene in the HepG2 cells, and underexpression of Kir6.2 gene decreased the proliferation and invasion of the HepG2 cells.

Cell Proliferation ; Genetic Vectors ; Hep G2 Cells ; Humans ; Neoplasm Invasiveness ; Plasmids ; Potassium Channels, Inwardly Rectifying ; genetics ; RNA, Small Interfering ; genetics

Endothelial injury, smooth muscle cells (SMCs) proliferation and migration are the same common pathophysiological processes of many cardiovascular diseases, such as atherosclerosis, hypertension, diabetes and restenosis. It is important to determine the functional interactions between endothelial cells (ECs) and SMCs under pathologic conditions. This work was to study the effects of ECs growth states on the proliferation and migration of vascular SMCs in cell coculture system. (3)H-TdR incorporation and flow cytometry were used to determine the effects of ECs growth states on the proliferation of SMCs. The number of migrating SMCs was counted. RT-PCR was used to analyze the expression of alpha-SM-actin mRNA. The results showed that (3)H-TdR incorporation decreased significantly from 14,900+/-1035 cpm/well in the control group to 8,575+/-749 cpm/well in the confluent ECs group (n=6, P<0.01), and increased to 27,268+/-2310 cpm/well in the proliferative ECs group ( n=6, P<0.01). The transition of SMCs from G(0)/G(1) phase to G(2)/M and S phases was blocked in the confluent ECs group but promoted in the proliferative ECs group. Compared with the control group, the number of migrating cells was about 4 times higher in the proliferative ECs group (n=6, P<0.01), while it in the confluent ECs group was only the half of the number of the control(n=6, P<0.05). The expression of alpha-SM-actin mRNA was increased significantly in the confluent ECs group(2.3+/-0.11 vs 1.4+/-0.12, P<0.05), but decreased significantly in the proliferative ECs group(0.92+/-0.08 vs 1.4+/-0.12, P<0.05). The results suggest that the biologic features of SMCs are influenced by ECs growth states. The proliferative ECs promote SMCs proliferation, migration and downregulate alpha-SM-actin mRNA expression significantly.
Actins ; metabolism ; Aorta ; cytology ; Cell Differentiation ; Cell Division ; Cell Movement ; Cells, Cultured ; Coculture Techniques ; Endothelium, Vascular ; cytology ; Epithelial Cells ; cytology ; Muscle, Smooth, Vascular ; cytology

OBJECTIVETo investigate the feasibility of computer simulation in maxillofacial firearm injury.

METHODSThe three-dimensional finite element models and simulations of 7.62 mm, 5.56 mm standard bullets projectile injuries to pig mandibular angle were established by using MIMICS, ANSA, LS-DYNA and LS-POST software. Based on the simulation results, the bullet hole diameters, energy loss values, energy loss rates, von Mises stress, effective strain, effective strain rate dynamic contours at different time points were used for biomechanical analysis.

RESULTSThe damage processe of 7.62 mm, 5.56 mm standard bullets projectile injury to pig mandibular angle were simulated successfully. The injury rate of 7.62 mm standard bullet and injury severity of the mandible were higher than that of 5.56 mm standard bullet.

CONCLUSIONSComputer simulation can simulate maxillofacial firearm injuries effectively and may become an important method for oral and maxillofacial firearm injuries analysis.

Animals ; Biomechanical Phenomena ; Computer Simulation ; Finite Element Analysis ; Imaging, Three-Dimensional ; Mandibular Injuries ; physiopathology ; Models, Biological ; Software ; Stress, Mechanical ; Swine ; Wounds, Gunshot ; physiopathology

OBJECTIVETo investigate the influnce of receptor activity modifying protein 1(RAMP-1) overexpression on enhancing effect of calcitonin gene-related peptide on MG-63 cells proliferation.

METHODSCultured MG-63 osteoblasts in exponential phase of growth were randomly divided into three groups: RAMP-1 overexpression group, empty vector control group and negative control group. RAMP-1 eukaryotic expression vector was constructed and stably transfected into MG-63 cells. Realtime-polymerase chain reaction, Western blotting and immunofluroescence were used respectively to detect the expression of calcitonin receptor-like receptor (CRLR) in the cells and its distribution on cell membrane. The status of proliferation was detected respectively at 0, 24, 48, 72, 96 h by cell counting kit-8 (CCK-8) and cells were collected to analyze their cycle respectively at 0, 8, 16, 24 h by flow cytometry.

RESULTSCRLR protein and mRNA expression levels of MG-63 cells in RAMP-1 overexpression group were significantly higher than the other two groups (P < 0.05). The A value of RAMP-1 overexpression group at 24, 48, 72, 96 h were 0.628 ± 0.175, 0.896 ± 0.592, 1.055 ± 0.004, 1.179 ± 0.618, respectively, which were significantly higher than that of the other two groups (P < 0.05). The difference was most pronounced at 72 h. S-phase fraction of RAMP-1 overexpression group was (1.25 ± 0.13)%, (68.79 ± 0.56)%, (64.49 ± 1.59)%, (57.82 ± 0.75)%, respectively, which were significantly higher than the other two groups (P < 0.05). The difference was most pronounced at 8 h.

CONCLUSIONSRAMP-1 overexpression can promote CRLR distribution on MG-63 cell membrane and enhance CGRP's promotion effect on MG-63 cell proliferation.

Bone Neoplasms ; metabolism ; pathology ; Calcitonin Gene-Related Peptide ; genetics ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Random Allocation ; Receptor Activity-Modifying Protein 1 ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the histological changes in the vestibular endorgans of Smad4 gene conditional knockout mice and to explore the influence of the Smad4 gene on vestibular development.

METHODSHistological changes of periphery vestibular organs in inner ear of Smad4 conditional knockout mice were investigated by frozen sections, immunofluorescence, confocal microscopy, scanning electron microscopy and transmission electron microscopy.

RESULTSThere was no Smad4 expression in the inner ear cartilage capsule of Smad4-/- mice. In Smad4+/- mice, Smad4 expression in the same cartilage capsule was positive, and it was strong positive in Smad4+/+ mice. Smad4 expression in vestibular sense epithelium, crista ampullaris and macula, was positive. And no difference was found among these three genotypes. Studying at scanning electron microscopy and transmission electron microscopy levels and anti-filament immunofluorescence showed that no pathological changes were observed in all the three genotype mice.

CONCLUSIONAlthough the Smad4 gene was knockout effectively in the auricular cartilage capsule of Smad4 conditional knockout mice,the histological changes of Smad4 conditional knockout mice in vestibulum auris internal were slightly.

Animals ; Ear, Inner ; anatomy & histology ; pathology ; Genotype ; Mice ; Mice, Knockout ; Smad4 Protein ; genetics ; Vestibule, Labyrinth ; anatomy & histology ; pathology

Carcinosarcoma ; chemistry ; pathology ; Female ; Humans ; Liver Neoplasms ; chemistry ; pathology ; Middle Aged

AIMTo detect the expression of ryanodine receptor (RyR) subtypes in normol rat airway smooth muscle cells(ASMCs) and changes during chronic asthma formation.

METHODSASMCs were cultured with collagen enzyme digestion method. The expression of subtypes of RyR were detected by RT-PCR. Purified PCR product linked with pGEM-T vector to make DNA sequence assay. Chronic asthma model was made with OVA, the changes of RyRs detected by RT-PCR.

RESULTSAll subtypes of RyR were expressed in airway smooth muscle cells of normol rat. The expression of RyR1 increased obviously compared with control group (P < 0.05) on chronic asthma.

CONCLUSIONCo-expression of three subtypes of RyR in ASMCs of normal rat, indicate that there are complicated intercellular Ca2+ regulation mechanism in ASM, moreover RyR1 might play a role during asthma development.

Animals ; Asthma ; metabolism ; Bronchi ; Male ; Myocytes, Smooth Muscle ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Ryanodine Receptor Calcium Release Channel ; genetics ; metabolism

Objective To observe the effect of Chaijin-Huayu decoction combined with endoscopic minimally invasive for gallbladder polyposis and its effect on quality of life.Methods A total of 91 patients with gallbladder polyposis were selected from March 2014 to early March 2017 outpatients and wards of Hebei Provincial Traditional Chinese Medicine Hospital.According to the method of random number table,the paitents were randomly divided into the observation group (n=46) and the control group (n=45).After three courses of treatment,the total effective rate,TCM symptom score and quality of life were observed in both groups,and the postoperative complications of the two groups were compared.Results After three courses of treatment,the total effective rate of the observation group was 91.3％ (42/46),while that of the control group was 88.9％ (40/45).There was no significant difference between the two groups (x2=0.149,P=0.592).In the observation group,the scores of subcardiac pycnosis and belching,right flank distended pain and burp were lower than those of the control group (t were 2.431,3.560 and 2.891 respectively,all P＜0.05).In the SF-36 scale,the scores of mental health,social function,physical pain,physiological function,emotional function,vitality,physiological function and overall health of the observation group were higher than those of the control group (t were 2.342,2.831,2.765,2.545,2.485,2.650,2.582,2.325 respectively,all P＜0.05);The incidence of surgical complications of the observation group was 2.2％ (1/46),while that of the control group was 17.8％ (8/45),and the difference between the two groups was statistically significant (x2=0.149,P＞0.05).Conclusions Chaijin-Huayu decoction combined with minimally invasive choledochoplasty could relieve symptoms,improve quality of life and reduce postoperative complications in patients with gallbladder polyposis.

BACKGROUNDPrimary total knee arthroplasty (TKA) in severe valgus knees may prove challenging, and choice of implant depends on the severity of the valgus deformity and the extent of soft-tissue release. The purpose of this study was to review 8 to 11 years (mean, 10 years) follow-up results of primary TKA for varient-III valgus knee deformity with use of different type implants.

METHODSBetween January 2002 and January 2005, 20 women and 12 men, aged 47 to 63 (mean, 57.19 ± 6.08) years old, with varient-III valgus knees underwent primary TKA. Of the 32 patients, 37 knees had varient-III deformities. Pie crusting was carefully performed with small, multiple inside-out incisions, bone resection balanced the knee in lieu of soft tissue releases that were not used in the series. Cruciate-retaining knees (Gemini MKII, Link Company, Germany) were used in 13 knees, Genesis II (Simth & Nephew Company, USA) in 14 knees, and hinged knee (Endo-Model Company, Germany) in 10 knees. In five patients with bilateral variant-III TKAs, three patients underwent 1-stage bilateral procedures, and two underwent 2-stage procedures. All implants were cemented and the patella was not resurfaced. The Hospital for Special Surgery (HSS) knee score was assessed. Patients were followed up from 8 to 11 years.

RESULTSThe mean HSS knee score were improved from 50.33 ± 11.60 to 90.06 ± 3.07 (P < 0.001). The mean tibiofemoral alignment were improved from valgus 32.72° ± 9.68° pre-operation to 4.89° ± 0.90° post-operation (P < 0.001). The mean range of motion were improved from 93.72° ± 23.69° pre-operation to 116.61 ± 16.29° post-operation (P < 0.001). No patients underwent revision. One patient underwent open reduction and internal fixation using femoral condylar plates for supracondylar femoral fractures secondary to a fall at three years. Three patients developed transient peroneal nerve palsies, which resolved within nine months. Two patients developed symptomatic deep vein thrombosis that was managed with rivaroxaban and thrombo-embolic deterrent stockings. There was no incidence of pulmonary embolism. Post-operative patient satisfaction was 80.7 ± 10.4 points in the groups. Prosthetic survival rate was 100% at mean 10 years postoperative.

CONCLUSIONSNot only hinged implants can be successfully used in variant-III valgus knees. As our results show, if proper ligament balancing techniques are used and proper ligament balance is attained, the knee may not require the use of a more constrained components. Our results also present alternative implant choices for severe knee deformities.

Arthroplasty, Replacement, Knee ; methods ; Female ; Humans ; Joint Deformities, Acquired ; surgery ; Knee Joint ; surgery ; Male ; Middle Aged

OBJECTIVEScreening and identification of differentially expressed genes in human primary hepatocellular carcinoma(HCC).

METHODSThe differentially expressed genes subtracted cDNA library of HCC constructed by suppression subtractive hybridization(SSH) technique was screened by colony in situ hybridization, then the positive clones were further screened with PCR amplification. The positive clones were sequenced and analyzed for homology in the Genbank databases with Basic Local Alignment Search Tool BLAST . The novel cDNA sequences were analyzed by Northern blot analysis.

RESULTSThirteen positive clones were obtained, and 11 cDNA sequences were identified. Sequences of 11 cDNA showed that 6 cDNA were homologous with the genes published in Genbank and 5 cDNA were unknown genes. Northern blot indicated that 3 novel cDNA(>300 bp) were only expressed in HCC.

CONCLUSIONThe subtracted cDNA library constructed by SSH technique contains differentially expressed genes of HCC. Three novel cDNA sequences might be differentially expressed genes of HCC. Further screening the library and gaining the whole gene sequence may lay a foundation for identifying differentially expressed genes in HCC.

Base Sequence ; Carcinoma, Hepatocellular ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Library ; Humans ; Liver Neoplasms ; genetics ; Molecular Sequence Data ; Sequence Analysis, DNA