Arthralgia ; diagnosis ; etiology ; therapy ; Humans ; Medicine, Chinese Traditional ; Syndrome ; Tendon Injuries ; diagnosis ; therapy

OBJECTIVETo explore a method for isolation and culture Dispase II and Trypsin-EDTA. Cells were seeded onto mitomycin C-treated of rat buccal mucosa stem cells and to identify the stem cells.

METHODSEpithelial cell mass were obtained by digesting rat buccal mucosa with 3T3 Swiss albino layer and cultured in DMEM for 24 hours, followed by K-SFM culturing. Some cells were induced to osteocytes and adipocytes and underwent ALP testing after 72 hours. Five days later, the primary cells were digested with trypsin and inoculated onto collagen IV-coated flasks and cultured at room temperature for 20 minutes. The adherent cells continued to be cultured with epithelial stem cell medium, then examined for identifying the clones, osteocytes, adipocytes, cytokeratin and ALP staining.

RESULTS83.96 percent of the primary epithelial cell mass were in G0/G1 phase by flow cytometry test. The clones were seen after 72 hours on 3T3 Swiss albino layer, and the osteocytes and adipocytes were positive. Cells were adhered quickly to collagen IV, in a shape of round or orbicular-ovate with strong refraction. The induced-osteocyte and adipocyte, cytokeratin and ALP were all positive.

CONCLUSIONSThe stem cell-like epithelial cells could be obtain using the 3T3 Swiss albino layer method. Sieved by collagen IV and cultured in epithelial stem cell medium could make the epithelial stem cells depurate and proliferate quickly.

Animals ; Cell Culture Techniques ; methods ; Cells, Cultured ; Mice ; Mice, Inbred Strains ; Mouth Mucosa ; cytology ; NIH 3T3 Cells ; Stem Cells ; cytology ; Tissue Engineering ; methods

OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection

Injury of the posterior pelvic ring can easily be caused by high-energy impact,and sacroiliac joint dislocation is the most common.The sacroiliac joint,as the hub of load transfer between the trunk and lower extremities,is essential to maintain the stability of the posterior pelvic ring,and once dislocation occurs,restoring the stability of the posterior pelvic ring by timely surgery is necessary.The current surgical approaches for the internal fixation of sacroiliac joint are mainly divided into anterior approach and posterior approach.The choice of the surgical approach directly affects the exposure of the surgical field,the stability of internal fixation and the prognosis of patients;therefore,it is particularly important to select the appropriate surgical approach and fixation method.In this paper,we briefly review the selection of sacroiliac joint fixation points,surgical approaches and postoperative complications.

OBJECTIVETo introduce a new method for enlarging the survival area of an expanded flap.

METHODSAfter the skin expander was inflated with enough injection, the first delaying was performed. In the operation, two incisions were made in the skin and subcutaneous tissue superficial to the expander capsule on both sides of the long axis of the expanded flap. After 10 to 14 days, the second delaying followed, in which one pedicle was divided to form a unilateral-pedicled, super-long random flap. When the flap was transferred to the recipient Since 2000,this technique has been used in 16 patients. All the area, the door site was closed directly.

RESULTSflaps survived completely.

CONCLUSIONSThe technique of twice delaying can enlarge the survival area of the expanded skin flap.

Adolescent ; Adult ; Child ; Cicatrix ; surgery ; Female ; Follow-Up Studies ; Humans ; Male ; Reconstructive Surgical Procedures ; methods ; Skin Transplantation ; methods ; Surgical Flaps

This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.
Cell Line ; Factor VIII ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

This study was purposed to constructe the three-plasmid system of the lentiviral vector carrying the green fluorescent protein (GFP) gene and to investigate the expression of GFP in T lymphocytes of the mouse. The polypurine tract (PPT) element, ubiquinone promoter (PUB) and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasmid pTK153. Human kidney 293T cells were co-transfected with the three-plasmid system containing packaging plasmid DeltaNRF, plasmid pTK153 and envelope plasmid VSV-G by using calcium phosphate DNA precipation and the expression of GFP was observed under fluorescence microscope after 12 hours. The viral particles were collected after transfection 72 hours, were frozen at -80 degrees C and were used to infect mouse T lymphocytes at multiplicity of infection (m.o.i.) of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). The results showed that the transfection efficacy was 63.04 +/- 7.24% in 293T cells analysed by FACS and the viral titer was (3.09 +/- 0.61) x 10(6) U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was (37.98 +/- 6.26)%. It is concluded that the three-plasmid system of lentiviral vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.
Animals ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; RNA, Viral ; analysis ; T-Lymphocytes ; metabolism ; Transduction, Genetic

OBJECTIVETo investigate the effect of bortezomib on prophylaxis of acute graft-versus-host disease (aGVHD) after mouse allogeneic-bone marrow transplantation (allo-BMT) and its mechanism.

METHODSC57BL/6 (H-2(b)) mice were used as donors and BALB/c (H-2d+) mice as recipients. After allo-BMT, the BALB/c mice were divided into 3 groups, ie. group A:BMT control, group B: BMT + early infusion of bortezomib (1 mg kg(-1) d(-1), day 0-3), group C: BMT + late infusion of bortezomib (1 mg kg(-1) d(-1), day 5-7). Clinical manifestations of aGVHD, pathohistological changes, survival rate and levels of recipients H-2(b) cells detected by flow cytometry in the recipient mice were observed. Monodirectional mixed lymphocyte culture (MLC) system was established ex vivo and different concentrations of bortezomib (0, 2, 4, 8 nmol/L) were added to the system. The viability of the cells was detected by CCK-8 assay and cells apoptosis by flow cytometry. The concentrations of IL-2, IFN-gamma, TNF-alpha in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe mice in group A developed typical aGVHD and all died of aGVHD within 3 weeks after transplantation, with a median survival time of (16.1 +/- 2.5) d. The symptoms of aGVHD was milder in group B than in group A, and the median survival time was significantly longer. The 60-day survival rate in group B was 70%, being significantly higher than that in other two groups(P<0.05). The mean value of donor-derived cell (H-2(b) cells) in group B was (98.1 +/- 1.1)% at 60 days. The symptoms of aGVHD was significantly severer in group C than in group A, and the median survival time was shorter. Bortezomib inhibited the cells viability in MLC system in a dose-dependent manner. After treated with 8 nmol/L bortezomib for 24 h, the inhibition ratio of cells viability was (41.4 +/- 6.0)%. The cell apoptosis rate increased gradually with bortezomib treatment for 12 h, 24 h and 36 h. After treated with 8 nmol/L bortezomib for 36 h, the apoptosis rate was (62.8 +/- 7.0)%. After treated for 24 h, the levels of IL-2, IFN-gamma and TNF-alpha in the supernatant were decreased.

CONCLUSIONSBortezomib administered immediately after allogeneic BMT can prevent aGVHD, improve the survival rate and have no influence of engraftment in the recipient mice. Delayed administration of bortezomib results in acceleration of aGVHD-induced mortality. Its mechanism maybe inhibition of the lymphocyte viability, increase of the cells apoptosis rate, and inhibition of secretion of IL-2, IFN-gamma, and TNF-alpha.

Animals ; Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; therapeutic use ; Bortezomib ; Cell Survival ; drug effects ; Cells, Cultured ; Disease Models, Animal ; Female ; Graft vs Host Disease ; prevention & control ; Interferon-gamma ; metabolism ; Interleukin-2 ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Pyrazines ; pharmacology ; therapeutic use ; Tumor Necrosis Factor-alpha ; metabolism

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.
Animals ; Dogs ; Factor VIII ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo explore the program of preoperative evaluation, preoperative preparation and separation operation of pygopagus conjoined twins.

METHODSClinical data of one case of pygopagus (male, gestational age 37 weeks, uterine-incision delivery, 3.5 months old; twins of triplets; total body weight 8.0 kg. The twins have self-governed extremities, anus, penis. Four limbs can move independently). Separated successfully were analyzed. The auxiliary examination of X-rays, MRI, ultrasound, CT and 3D reconstruction, sensory evoked potential showed that there were process hypoplasia and lamina bifid of lumbosacral vertebrae below L3. Dural sac connected below L3 and conus medullaris located at L3. There were no conjunction of spinal cord and cauda equine. The decision of direct suture of dural sac was made by preoperative evaluation through measurement of circumference and area of conjoined dural sac. The separation surgery program was decided through team debate and sham operation. Separation operation was performed under the general anaesthesia. Crossing V-shaped skin flap was used to cover the wound surface. The dura of conjoined twins were sutured directly. The tension of skin flap was normal postoperative with continuing negative pressure drainage under the flap and pressure sterilized dressing.

RESULTSSuccessful separation of pygopagus conjoined twins was achieved. Lower extremities movements of separated twins were normal 6 h after operation. The drainage was removed 1 day postoperative, blood supplies of skin flap were normal. There were no complications of infection, cerebrospinal fluid leakage and neurological deficit. The healing of the skin flap was good. With 6 months follow-up, the growth and neurological function were normal.

CONCLUSIONConsummate preoperative preparation, accurate preoperative investigations, meticulous operative management, careful postoperative administration and good team cooperation are the keys to successful separation of pygopagus conjoined twins.

Buttocks ; Follow-Up Studies ; Humans ; Imaging, Three-Dimensional ; Infant ; Male ; Prognosis ; Surgery, Computer-Assisted ; Surgical Flaps ; Treatment Outcome ; Twins, Conjoined ; surgery