AIM:To investigate the effect of calcitonin gene-related peptide (CGRP)transfection into c-kitpos cardiac stem cells (c-kit+CSCs) on the cell viability.METHODS: Under the sterile condition, the auricles of SD rats were taken out , and then c-kit +CSCs were collected through enzyme digestion and immunomagnetic bead separation (MACS).The cells were identified by flow cytometry .c-kit +CSCs were transfected with enhanced green fluorescent pro-tein CGRP lentiviral vector ( Lv-EGFP-CGRP) or enhanced green fluorescent protein lentiviral vector ( Lv-EGFP).The cells were randomly divided into Lv-EGFP-CGRP-CSCs group , Lv-EGFP-CSCs group and CSCs group .The transfection was observed under the fluorescence microscope .The transfection efficiency was detected by flow cytometry .The CGRP protein secretion in the cell culture supernatants was detected by ELISA .The viability of c-kit+CSCs transfected with Lv-EGFP-CGRP or Lv-EGFP was measured by CCK-8 assay.RESULTS:c-kit+CSCs were isolated and cultured successfully .The expression positive rate of c-kit was 91.0%and the expression positive rates of CD 45 and CD34 were 4.5% and 4.0%, respectively.After transfected with lentivirus for 48 h, the stable fluorescence in c-kit +CSCs was observed under fluores-cence microscope .The transfection efficiency were 80%when MOI was 20.The level of CGRP was significantly increased in Lv-ECFP-CGRP-CSCs group compared with Lv-EGFP-CSCs group and CSCs group (P<0.05).Meanwhile, transfection with lentiviral vector in each group did not affect the viability of c-kit+CSCs.CONCLUSION:Transfection of Lv-EGFP-CGRP into c-kit+CSCs was successful .The secretion of CGRP was found in the transfected c-kit+CSCs and the viability was not changed after transfection .CGRP-modified c-kit+CSCs may play a role in treating myocardial infarction .

A real-time polymerase chain reaction ( PCR ) micro total analysis system (μ-TAS ) integrated nucleic acid extraction, PCR amplification and real-time-fluorescent PCR detection on a same microfluidic chip was prepared for the fully automated and on-chip analysis of nucleic acid. The proposed method had the advantage such as low sample consumption, fast analysis and simple operation and so on. Micromachining technology was used to fabricate the anodic molds of integrated nucleic acid extraction microfluidic chip. A polydimethylsiloxane (PDMS) substrate with 3D channels was manufactured by a combination of molds and an injection molding method. The glass substrate and the chip were bonded together using a plasma treatment. The μ-TAS included a microfluidic control device whose micro fluidic velocity ( 0-10 mL/min ) could be adjusted, a TEC platform which the precision of temperature control was 0. 1℃ and a CCD detection module. The DNA of human blood was extracted by using a silica gel membrane method on the microfluidic chip. The processes of DNA extraction and detection were preset in the μ-TAS. Human blood lysate ( 20 μL ) were driven to the extraction chamber and was then washed. The fluidic drive speed was 2 mL/min. DNA and PCR reagents were mixed and then were driven into the PCR chamber. The fluidic drive speed was 1 mL/min. The GAPDH gene in extracted genome DNA was amplified by PCR and detected. The amplified product was verified by melting analysis. The results of nucleic acid extraction method on the chip were compared to those obtained using a standard manual centrifuge extraction method. The amplification curves were obvious. Ct values of the chip method were 25 . 3 and 26 . 9 . The denaturation temperature of all the melting was 89 . 9 ℃. The results validated that the chip-based method and device realized the extraction of nucleic acid, amplification and detection automatically.

AIMTo determine the molecular weight and first-order structure of somatostatin.

METHODSThe molecular weight of somatostatin was determined by electrospray ionization mass spectrometry. Somatostatin was deoxidized by 2-mercaptoethanol. A series of typical fragment ions of deoxidized product were obtained by insource collision-induced dissociation (CID).

RESULTSThe m/z of quasi-molecular ion [M + H]+ of somatostatin was 1,637.8 and [M + Na]+ was 1,659.5. The m/z of double-charge ion [M + 2H]2+ was 819.5 and [M + H + Na]2+ was 830.3. It showed that the molecular weight of somatostatin was 1,636.7. The y and b series of fragment ions of deoxidized product were obtained by adjusting the fragmentor voltage. It was determined that the first-order structure of deoxidized product of somatostatin was A-G-C-K-N-F-F-W-K-T-F-T-S-C.

CONCLUSIONThe molecular weight and first-order structure of somatostatin were confirmed.

Amino Acid Sequence ; Molecular Structure ; Molecular Weight ; Somatostatin ; analysis ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods

OBJECTIVETo establish a set of suitable and reliable methods for HBV genotyping and to study the distribution of HBV genotypes.

METHODSType-specific nucleotides were searched through alignment of S genes (more than 1000 sequences) listed in GenBank. Then, type-specific primers were designed and type-specific primer PCR was used to genotype the 238 HBV strains. S genes of the untyped strains were further amplified and sequenced to find out their genotypes with type-specific nucleotide analysis.

RESULTSAll the 238 HBV strains were genotyped. 159 (66.8%) cases were genotype B, 69 (28.9%) were genotype C, 6 (2.5%) were mixtures of genotypes B and C and 4 (1.6%) were mixtures of genotypes B and D. No genotypes of A, E, F, G, and H were found.

CONCLUSIONGenotypes B and C are the most common types for HBV strains. Mixtures of genotypes B and C or genotypes B and D coinfection rarely existed. There is no relationship between the gender of the patients and HBV genotypes (X2 = 0.794, P more than 0.05).

DNA Primers ; DNA, Viral ; blood ; genetics ; Female ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Nucleotides ; genetics ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

Objective To explore the importance and methods of retaining articularis during pri- mary total hip arthroplasty(THA)and reconstruct soft tissue balance of hip joint after THA.Methods From February 2003 to August 2005,41 eases(43 hips)including 19 males and 22 females at age of 46- 80 years(mean 66.5 years)were treated with THA with retained capsule(Group R)and other 42 cases (44 hips)including 20 males and 22 females at age of 43-80 years(mean 64.3 years)with standard THA (Group S).Preoperative diagnosis found femoral neck fractures(GardenⅢⅣ)in 13 cases(13 hips)in Group R and 14(14 hips)in Group S;acetabular dysplasia(CroweⅢ)in 9(9 hips)in Group R and 8 (hips)in Group S;Osteoarthritis in 6(8 hips)in Group R and 7(8 hips)in Group S;and femoral head osteonecrosis(FicatⅢⅣ)in 13(13 hips)in Group R and 13(14 hips)in Group S.There were 13 hips of cement prostheses in Group R and 11 in Group S,8 cementless prostheses in Group R and 8 in Group S, 22 cement and cementless prostheses in Group R and 23 in Group S.Gibson's approach was used in both groups.Group R used the method of retaining capsule and little supination muscles during the operation to reconstruct responsibly soft tissue balancing of postoperation for THA.For comparison,Group S used the method of standard which resected a lots of capsule and didn't reconstruct it.The comparative items between Group R and Group S included incisional length,operative time,operative bleeding,drainage transfusion, infection,dislocation,postoperation standing,postoperation walking and Harris's score.Results All cases in Group R and Group S were followed for 6-22 months(mean 16.5 months in Group R and 16.7 months in Group S).There was significantly statistical difference upon interoperative and postoperative data between Group R and Group S.The result of Group R was significantly better than that of GS.Conclu- sion Retaining articularis during primary THA can minimize operative trauma,reconstruct soft tissue bal- ance and augment hip stability to get postoperative functional recovery.

Objective To explore the significance of ADC value combined with MRI signs on identifying benign and malignant breast lump lesions with the type of TICⅡ.Methods 187 patients with breast lump lesions of TICⅡ,which were confirmed by surgical pathology,proceeded MRI.The ADC value,early-phase enhancement rate,length of lesions,lobulated appearance and spiculation, inverted nipple,thickening of the skin and the length of ipsilateral axillary lymph nodes were analyzed and recorded.T-tests was used to analyzed the measurement data,χ2test was used to analysis the attribute data.The ROC curves were used to evaluate the diagnostic efficiency of ADC value and MRI signs.Results The ADC value was (1.418±0.299)×10-3mm2/s and(0.860±0.142)× 10-3mm2/s (P<0.01)for breast benign and malignant lesions respectively,while the early-phase enhancement rate were (170.387± 74.580)% and (160.778±39.786)%(P=0.258)respectively.When ADC values were used for differential diagnosis of breast benign and malignant lesions with a threshold of 1.017×10-3mm2/s,the area under the sensitivity and specificity were 89.0% and 98.0% The sensitivity and specificity of lobulated appearance,spiculation,inverted nipple and thickening of the skin were 92.6% and 33.3%, 88.2% and 88.2%,20.5% and 94.1%,35.3% and 88.2%,respectively.When the 1.0 cm was used as the threshold of the length of ipsilateral axillary lymph,its sensitivity and specificity were 47.8% and 80.4%.The ROC curve of early-phase enhancement rate showed no diagnostic capability(P>0.05).Conclusion ADC value combined with MRI features are helpful to improve the sensitivity and specificity in breast lesions with the type of TICⅡ.

Objective To investigate the clinical effectivity of the muscular flap transposition and induced membrane technique in the emergency treatment for the limb salvage of Gustilo type Ⅲ B/C open fracture of lower leg. Methods From July, 2015 to December, 2017, 10 cases of Gustilo type Ⅲ B/C fracture of lower leg with bone defects were performed limb salvage surgery. Induced membrane technique was used to fill the bone defects in the emergency room.The gastrocnemius and/or soleus muscular flaps were transposed to cover the bone cement or ex-posed bone simultaneously in emergence treatment. After the wound healed completely, traditional bone grafting was used to repair the bone defects. There were 4 cases of Gustilo type Ⅲ B and 6 cases of Gustilo type Ⅲ C. The aver-age length of bone defect was (5.25±1.70) cm ranging from 3.0 cm to 11.0 cm. The gastrocnemius medial head flaps were performed in 5 cases, the combined application with the gastrocnemius medial head flaps and the medial hemimuscular flaps of soleus were performed in 2 cases, and medial hemimuscular flaps of soleus were transposed in 3 cases. Results The wounds in 6 cases were healed at one stage, but 2 cases healed by dressing because the exudate after skin grafting.In 1 case, the cross-leg flap was used to cover the exposed bone cement due to the necro-sis of soleus flap. The other 1 was performed the transposition of the lateral gastrocnemius flaps because the exposure of bone cement after the necrosis of the upper and lateral muscles in lower leg. In the second-stage, the bone defects were reconstructed by traditional bone grafting. The average healed time of bone was 7.2 months ranging from 5 months to 9 months. At the last followed-up time, all patients recovered their function of weight-bearing. The Paley's score of the adjacent joints: excellent in 8 cases and good in 2 cases. Conclusion The combination with induced membrane technique and local muscular flap transposition in emergency surgery is an effective method to limb salvage for the Gustilo type Ⅲ B/C open fracture of lower leg.

Objective:To screen and identify the protein that interacts with the adhesion protein of Mycoplasma genitalium (MgPa)from T7-phage display cDNA library of human uroepithelial cells(SV-HUC-1).Methods:Recombinant adhesion protein of My-coplasma genitalium(rMgPa)was used as target molecule to biopan the T7 phage display cDNA library of SV-HUC-1 cell,the selected positive clones were analysed using DNA sequencing and BLAST analysis and identified by means of indirect ELISA,Dot immunoblot and Far-western blot.Results:After four rounds of biopanning,positive phages were obviously enriched.According to the results of DNA sequencing and BLAST analysis,the selected randomly 32 positive clones included 7 kinds different sequences,of which the number of RPL35 repeats was the most.The results of indirect ELISA,Dot immunoblot and Far-western blot showed that 7 representative phages could bind specifically with rMgPa.Conclusion:60S ribosomal protein L35(RPL35) may be the interacting protein of MgPa,which lays the experimental foundation for understanding the function of MgPa and the pathogenesis of Mycoplasma genitalium.

Objective: To compare the effect of galectin-3 (Gal-3), NT-proBNP and echocardiography paramerters on assessing cardiac function in patients with chronic heart failure (HF). Methods: A total of 144 patients treated in our hospital from 2016-03 to 2016-11 were enrolled. According to the NYHA classification, the patients were divided into 2 groups: HF group and Normal cardiac function group. n=72 in each group. Basic clinical information was collected, blood levels of Gal-3 and NT-proBNP were examined, echocardiography was conducted to measure left ventricular ejection fraction (LVEF) and left ventricular end diastolic diameter (LVEDD). Correlations between Gal-3, NT-proBNP and echocardiography parameters were studied, the abilities of Gal-3, NT-proBNP and echocardiography for estimating HF were compared. Results: Compared with Normal cardiac function group, HF group had increased blood levels of NT-proBNP [3499.5 (1431.3-9088.0) ng/L] vs [384.1 (122.1-1540.5) ng/L] and Gal-3 [3.0 (1.71-5.8) pg/ml] vs [1.9 (1.4-2.6) pg/ml], decreased LVEF [49.5% (42%-58%)] vs [62.5% (59%-67%)], enlarged LVEDD [52.0 (46.3-57.8) mm] vs [46.0 (42.0-49.0) mm] and elevated serum creatinine [113.6 (90.5-152.7) umol/L] vs 82.4 (69.1-97.4) umol/L], all P<0.05. Correlation analysis showed that NT-proBNP and Galectin-3 were positively related to LVEF and LVEDD; Gal-3 and NT-ProBNP had the strongest correlation (r=0.57, P<0.01). The AUC of ROC for Gal-3 was 0.674 (0.584-0.763), for NT-proBNP was 0.837 (0.771-0.902) and for LVEF was 0.806, (0.735-0.878) which implied that NT-proBNP was the most powerful parameter for estimating HF. Conclusion: Gal-3 had the ability to estimate HF and could be used as a biomarker, while its ability was lower than NT-proBNP in clinical practice.

OBJECTIVETo explore a new method of establishing HepG2 cell model of steatosis and observe the expression and significance of nuclear factor erythroid-2p45-related factor 2(Nrf2)/antioxidative response element (ARE) pathway related factors in HepG2 cells of steatosis.

METHODSHepG2 cells were induced with DMEM containing 25% fetal bovine serum, 0.1% MCT/LCT Fat Emulsion and 0.1 mmol/L free fatty acid (FFA) at different stages and the control group cells were cultured with normal DMEM medium. After the cell models were successfully established, lipid droplets in cytoplasm were observed with Oil Red 0 staining, and the triglyceride (TG) accumulation in HepG2 cells were tested by biochemical assay. Intracellular reactive oxygen species (ROS) concentration were detected by flow cytometry. Nitric oxide (NO), superoxide dismutase(SOD), malonyldialdehyde(MDA) and glutathione peroxidase(GSH-Px) were tested by biological reagent kit, while the protein expression of nuclear factor erythroid-2p45-related factor 2(Nrf2), heme oxygenase-1 (HO-1) and

NAD(P)Hquinone oxidoreductase-1(NQO1) were analyzed by Western blot.

RESULTSCompared with that in the control group, red cytoplasmic lipid droplets were visible in model group; TG,ROS, NO, MDA concentration (P < 0.05, P < 0.01) and the protein expression of Nrf2, HO-1 and NQO1 (P < 0.05, P < 0.01)were significantly higher in model group, while SOD, GSH-Px concentration reduced significantly (P < 0.01).

CONCLUSIONThe in vitro cell model of steatosis and oxidative stress was successfully established. The activation of Nrf2/ARE pathway related factors maybe relevant to the overreaction of oxidative stress in HepG2 cells of steatosis.

Antioxidant Response Elements ; Culture Media ; Fatty Acids, Nonesterified ; Fatty Liver ; metabolism ; GA-Binding Protein Transcription Factor ; Glutathione Peroxidase ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hep G2 Cells ; Humans ; Malondialdehyde ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Nitric Oxide ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Triglycerides ; metabolism