We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection

OBJECTIVETo seek a new method for reconstructing bilateral intemrnal jugular vein invaded by metastasis lymph node in advanced oral cancer patients.

METHODSA combination of microvascular anastomosis and longitudinal constriction suture venoplasty was performed to reconstruct internal jugular vein. We resected the part of the bilateral internal jugular vein of advanced oral cancer patients invaded by metastasis lymph node and used the external carotid vein to reconstruct the internal jugular vein. A part of the vessel wall of the internal jugular vein could also be resected to reconstruct the vein. Longitudinal constriction suture venoplasty could slowly narrow the lumen diameter of the internal jugular vein. Thus, difference in anastomosis diameter should be avoided because it generates eddy currents and subsequently causes blood clots. A total of five advanced cases of oral squamous cell carcinoma were involved in this study. We performed bilateral radical neck dissection on all patients to reconstruct the internal jugular vein and observed their postoperative conditions.

RESULTSPostopera-tive follow-up of 5 months to 19 months was performed on all patients. Doppler or CT angiography and related tests showed no internal jugular vein thrombosis. No patient with facial edema, throat swelling, cerebral edema, and high intracranial pressure or other serious complications caused by blocked venous blood was observed. The one-year survival rate of five patients was 60% (3/5).

CONCLUSIONMicrovascular anastomosis combined with longitudinal constriction suture venoplasty is a new method for reconstructing internal jugular vein. This method was proved successful and clinically feasible.

Carcinoma, Squamous Cell ; Constriction ; Humans ; Jugular Veins ; Lymphatic Metastasis ; Mouth Neoplasms ; Neck Dissection ; Postoperative Period ; Reconstructive Surgical Procedures ; Sutures

Objective To explore the bacterial drug resistance in blood culture in 2015 from the members of Antimicrobial Re-sistant Investigation Net of Shaanxi province,and to guide the clinicians touse antimicrobial drugs rationally.Methods All the objective bacterial isolates were collected and identifiedsusceptibility date by software WHONET 5.6.Results 6 871 bacterial isolates and their antibacterial susceptibilitydata were collected which included 3 199 (46.56%)Gram-negative bac-terial isolates and 3 672 (53.44%)Gram-positivebacterial isolates.The top five populationsof Gram-positive bacterial iso-lates were Staphylococcus epidermidis (30.94%),Staphylococcus hominis (17.84%),Staphylococcus haemolyticus (11.74%),Staphylococcusaureus (9.69%)and Enterococcus feacium (6.29%).The top five populationsof Gram-negative bacterial isolates were E.coli (43.67%),Stenotrophomonas maltophilia (14.63%),K.pneumoniae (13.47%), P.aeruginosa (4.13%)and Acinetobacter baumannii (3.63%).Theisolating rates of methicillin-resistant Staphylococcus aureus (MRSA)and methicillin-resistant coagulase negative Staphylococcus (MRCNS)were 31.2% and 76.1%,respec-tively.No vancomycin resistant Staphylococcusisolates were found.There were 0.9% E.faecium vancomycin-resistant iso-lates.The isolates of Enterobacteriaceae were still highly susceptible to carbapenem,whosetotal resistance rate was below 4.0%.The resistance rates of A.baumannii to most surveillance drugs in cludingimipenem were above 50.0% and the iso-lates of P.aeruginosa were still highly susceptible to most surveillancedrugs.Conclusion It is severe that the situation of bacterial drug resistance in blood culture in Shaanxi province.Should fullyuse bacterial drug resistance surveillance results for supervision and administration,and take effective measures forcontrolling the spread of resistant isolates.

Objective To study the value of CT on defining the gross tumor volume (GTV) in three-dimensional conformal radiotherapy (3DCRT) and intensity-modulated radiation therapy(IMRT), by comparing the GTV contoured on preoperative CT with postoperative pathology guided GTV in patients with non-small cell lung cancer (NSCLC). Methods Thirty-three patients with histologically proved NSCLC had had CT scan of the thorax before sugery. We contoured the GTV for both primary tumor (GTV-T) and the regional metastatic lymph nodes (GTV-N) on preoperative CT scan. The GTV-T was defined on lung window while the GTV-N on mediastinal window. The metastatic lymph node was defined as≥ 10mm in diameter of short axis,while

Objective To investigate hemodynamic changes of deep vein in lower limb during the perioperative period of total hip arthroplasty (THA) and early diagnosis of deep vein thrombosis (DVT). Methods Doppler ultrasound, hemorheology detection and plasma D-dimer testing were done on 62 patients treated with THA. Statistical analysis was carried out on the data of patients with or without DVT to study the early diagnosis of DVT. Results The results of Doppler ultrasound showed DVT in 8 patients. Compared with postoperative concentration of plasma D-dimer, the preoperative con-centration of plasma D-dimer was significandy higher in patients with or without DVT (P < 0.05). The levels of hemorheological indices were significantly increased at postoperative day 7 (P < 0.05). Con-clusions Doppler ultrasound combined with plasma D-dimer testing and hemorheology detection are helpful in early diagnosis of DVT.

Carcinoma, Hepatocellular ; microbiology ; pathology ; Cell Line, Tumor ; Cyclin D1 ; biosynthesis ; genetics ; Helicobacter pylori ; physiology ; Humans ; Liver Neoplasms ; microbiology ; pathology ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo explore whether bone marrow stem cells (MSCs) from adult mice can be induced to differentiate into hepatocytes by hepatocyte growth factor (HGF) alone and the time phase characteristics in the differentiation progress.

METHODSAdult mouse MSCs were treated with or without 100 ng/ml HGF, on days 0, 7, 14, 21, and 28. The morphologic characteristics of the cells were examined; the albumin (ALB), AFP mRNA was analyzed sub-quantively using reverse transcription polymerase chain reaction (RT-PCR) and immumohistochemistry techniques. The expression of ALB, AFP and CK19 were detected by using anti-ALB, AFP and CK19 antibodies.

RESULTSFreshly isolated adult mouse MSCs expressed ALB and AFP mRNA weakly; in the group without HGF, no ALB mRNA was detected on day 7. The expression of AFP mRNA was reduced significantly on day 7, and could not be detected anymore after day 14. In the HGF treated group, ALB mRNA was not detected on day 7, but the positive lane appeared again on day 14, and the expression of ALB mRNA was increased on day 21 but reduced in the following days. The AFP mRNA was positive at all times, however it tended to decrease after day 14 in the HGF treated groups. The result of immumohistochemistry was consistent with that of RT-PCR, and CK19 was always negative.

CONCLUSIONAdult mouse MSCs can be induced into hepatocyte differentiation in vitro. The optimal time for the induction was 2 to 3 weeks.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; Male ; Mice ; Stem Cells ; cytology ; Time Factors

OBJECTIVETo observe the effect of electroacupuncture at Zusanli (ST 36) on phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the dorsal horn of spinal cord induced by plantar inflammation in the rat.

METHODSAll the rats were randomly divided into 5 groups: normal control group, simple electroacupuncture group, formalin group, formalin plus ipsilateral electroacupuncture group and formalin plus contralateral electroacupuncture group. The acute inflammation animal model was made by injection of 100 microL of 4% formalin into the right posterior foot pad. Electroacupuncture was given at "Zusanli" (ST 36) for 30 min, with sparse-dense waves, frequency 2-15 Hz, and intensity 2-3 mA. One and a half hours latter, the rats were killed under anesthesia, and pERK1/2 expression in the lumbar dorsal horn were investigated with immunohistochemical method.

RESULTSThe positive cells were rarely seen (6.45 +/- 1.05) in the superficial spinal cord in the control group; a few cells (14.07 +/- 3.19) in ipsilateral superficial spinal cord were found in the electroacupuncture group. The number of pERK1/2-positive neurons (26.57 +/- 4.93) in lamina I - II0 of the ipsilateral dorsal horn in the formalin group increased significantly. After electroacupuncture at ipsilateral Zusanli (ST 36), the number of positive cells (20.79 +/- 5.21) had a tendency to decrease, but with no statistically significant difference. However, after electroacupuncture at contralateral Zusanli (ST 36), the number of positive cells (14.75 +/- 3.03) significantly decreased as compared with the non-acupuncture group (P < 0.05).

CONCLUSIONThe inhibition of ERK1/2 phosphorylation in the spinal cord dorsal horn by electroacupuncture is possibly involved in acupuncture analgesic effect.

Acupuncture Analgesia ; Acupuncture Points ; Animals ; Electroacupuncture ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Posterior Horn Cells ; enzymology ; Rats ; Rats, Sprague-Dawley

Objective: To observe the effects of sodium dimercaptosulphonate (DMPS) plus Gandou tablet, DMPS and calcium disodium ethylene diaminotetraacetate (EDTA) on improving liver cirrhosis and liver function of hepatolenticular degeneration (HLD) patients. Methods: One hundred and forty-six HLD patients were divided into A, B, C three groups, and treated with DMPS plus Gandou tablet, DMPS and EDTA respectively, the therapeutic course was 8 weeks for three groups. The ultrasonography of liver, electrophoresis of serum protein and excretion of urinary copper were observed. Results: The ultrasonography of liver was improved in all groups, the rate of improvement of group A was 54.0%, B was 44.0% and C was 39.1%. The amounts of serum albumin in group A and B increased (P＜0.01，P＜0.05), and γ-globulin decreased in all groups (P＜0.05). The excretion of urinary copper increased obviously in all groups (P＜0.01), and group A,B increased more than that of group C (P＜0.05). Conclusions: The de-copper therapy could improve liver cirrhosis and liver function. The effect of DMPS plus Gandou tablet was better than that of DMPS and EDTA.

OBJECTIVETo explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein.

METHODSFull length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSThe mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium.

CONCLUSIONSIt has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.

Cell Line ; Genetic Engineering ; Genetic Vectors ; Hepatitis B virus ; genetics ; Mutation ; Plasmids ; RNA, Antisense ; physiology ; Transfection ; Viral Core Proteins ; physiology ; Virus Assembly ; Virus Replication