Objective To investigate the role of basic fibroblast growth factor（bFGF）on the vascularization of bone matrix gelatin（BMG） embedded vascular bundles in femoral anteriomedialis in the rabbits.Methods A longitudinal incision was done at the hyper-knee joint of femoral anteriomedialis in the Japan big ears white rabbit.Thirty six rabbits were divided into group A（n =12,arteria saphena and vena saphena were liberated and embeded to the groove of BMG soaked by 2 μg/ml bFGF0,group B（n =12,arteria saphena and vena saphena were liberated and embeded to the groove of BMG untreated）,and group C（n =12,the BMG untreated were directly implanted）.The rabbits were sacrificed at 4 weeks,8 weeks and 12 weeks after the surgery respectively by perfusing ink.After the implants were dislodged,transparent specimens were made and Masson stained for histological observations and quantitative analysis.Results After 12 weeks of operation,the neovascularization arranged in an ordered manner in group A,gradually trended to be orderly in group B,and were cluttered mainly on the edge of the implants in group C.The osteogenic and neovascularization areas of group A were the largest on each time point.Conclusion bFGF could promote the vascularization of BMG embedded vascular bundle.There was a positive correlation between osteogenesis and vascularization.

Objective To explore the relationship between restoring Gissane angle and prognosis in calcaneal fracture surgery. Methods Forty patients with single side calcaneal fracture (SandersⅡ-Ⅲ), Gissane angle changed more than 15°and having performed open reduction and internal fixation with steel plate were enrolled. In them, 25 patients (experimental group) recovered Gissane angle in surgery referencing the healthy side with X-rays. Another 15 patients (control group) didn't recover Gissane angle. The ratio of calcaneum height and length was measured at 1 week after surgery. At 6 and 12 months after surgery, the function were valued by Maryland score. Results The ratio of calcaneum height and length in experimental group was significantly higher than that in control group:0.60± 0.04 vs. 0.55±0.05, and there was statistical difference (P<0.05). All the patients were followed up. At 6 and 12 months after surgery, the Maryland score in experimental group were significantly higher than those in control group:(88.9± 5.7) scores vs. (80.5±7.3) scores and (89.5 ±5.5) scores vs. (82.5 ±6.4) scores, and there were statistical differences (P<0.05). Conclusions Restoring Gissane angle is benefitial for prognosis. So in calcaneal fracture surgery, the Gissane angle should be recovered as much as possible referencing the healthy side.

BACKGROUND:Genotype of Kunming mice was similar to human population,thus an establishment of embryonic stem cell line is beneficial for research of transgenic animal.However,the best time to collect embryo has been less reported yet.OBJECTIVE:To find the best time to collect embryos from Kunming mice.METHODS:The embryos were collected from mother mice of 2.5,3.5,and 4.5 pregnant days.Microscope was used to evaluate the growth condition of embryos,embryo attaching rate(A/C),inner cell mass(ICM)growing rate(I/C),embryonic stem cells (ESCs)clone growing rate(P1/C)and ESCs subclone growing rate(P2/C).The cells were then stained with alkaline phosphatase.RESULTS AND CONCLUSION:Most of the 2.5-pregnant-day embryos were 16-cell-phase embryos.The 3.5-pregnant-day embryos were morulas while the 4.5-pregnant-day embryos were blastulas.There were no significant differences in A/C,I/C,P1/C,P2/C between 2.5 and 3.5 pregnant days(P ＞ 0.05).The 4.5-pregnant-day indicators mentioned above were significantly greater than those two groups;therefore,4.5-pregnant-day embryos were the best source to culture,clone,isolate and passage ESCs.

BACKGROUND: Anterior and dorsal root nerve tracts should be separated to small tracts in high selective dorsal rhizotomy, because detailed tract separation will benefit electrostimulation, thereby helping correctly cutting the lower-threshold Ia nerve fibers that cause convulsion, and meanwhile sensory nerve fibers in dorsal nerve root can be reserved as many as possible.OBJECTIVE: To meet the needs of limited and high selective spinal dorsal rhizotomy, anterior and dorsal root of spinal nerve were microanatomized to be certain of the separation standard and the number of small nerve tracts, so as to provide reliable basis and novel operative standard for clinical operation.DESIGN: Single sample experiment with adult corpses as subjects.SETTING: Orthopedic Department of the First Affiliated Hospital and Department of Anatomy, Jinzhou Medical College.PARTICIPANTS: This study was carried out at the Anatomical Laboratory of Jinzhou Medical College in December 1999. Fifteen adult corpses, 11 males and 4 males, were donated, and the donators signed informed consent when alive.tained from the 15 adult spinal cords (30 sides) and subjected to morphoanterior and dorsal roots of L5 spinal cord were obtained from a fresh corpse for immunohistochemical staining. The starting part, middle part and the exterior of intervertebral foremen was cut into slices, and the total number of nerve fibers, the number of Ia nerve fibers responsible for convulsion, and their percentage in the total fibers were counted. Meanwhile the distribution and the number of Ia nerve fibers in the three parts were compared.ber of nerve fibers per 100 μm2, the percentage of Ia nerve fibers in the total nerve fibers at the starting part, middle part and exterior of intervertebral foremen of spinal nerve dorsal root.root filaments. Microsurgical observation proved that dorsal root could be divided into 10-18 small tracts and anterior root 6-11 tracts; the diameter number of nerve fibers in the three parts of spinal nerve dorsal root was (3 243±143) fibers per 100 μm2, including (1 702±85) Ia nerve fibers that constituted about 52.5% of the total nerve fibers. Ia nerve fibers were found to be evenly distributed in dorsal root and no gathering could be observed.CONCLUSION: The tracts in anterior and dorsal roots of spinal cord should be separated as minutely as possible during improved dorsal rhizotomy, which is beneficial for cutting off Ia nerve fibers correctly. Generally,the anterior root consists of 6-11 small tracts and dorsal roots of 10-18small tracts, and nerve tracts should not be cut off over 1/2 of total dorsal nerve fibers.

Objective To investigate the effect of construct the Notch1 (NICD) eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Rat BMSCs were experimented as the object. NICD eukaryotic expression vector was constructed. pEGFP-N1-NICD expressing plasmids were used to transfect BMSCs. The study included control group (CON group), empty vector group (VEC group) and the trans-fection group (TRA group). After 48-hour transfection, BMSCs were observed for general morphology. The protein expres-sions of NSE, GFAP and Notch1 were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy-cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay. Results The DNA sequencing con-firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after 48-hour transfection. The relative expression levels of Notch1 and GFAP mRNA and pro-tein were significantly higher in TRA group than those in VEC group and CON group (P<0.05), and there was no significant difference between VEC group and CON group. After 48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group (P<0.05). The late apoptotic rate was significantly higher in VEC group than that of CON group. The proportion of G1/G0 cells was significantly higher in TRA group than that of CON group and VEC group, but S and G2/M cells were significantly lower (P<0.05). The value of growth curve was gradually de-creased in TRA group than that of CON group and VEC group (P<0.05). Conclusion The high expression of NICD gene might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentiation.

Objective To investigate the effects of Ginsenoside Rg1 on the expression of nitric oxide synthase (NOS) in brain tissue after cerebral arterial thrombosis in adult rats. Methods Thirty rats were randomly divided into the sham-operative group, cerebral ischemia-reperfusion group, Ginsenoside Rg1-L group, Ginsenoside Rg1-M group, Ginsen-oside Rg1-H group and nimodipine group (n=5 for each group). The ischemia-reperfusion rat model was established by mid-dle cerebral artery occlusion. The neurological score after reperfusion was observed. The levels of nitric oxide (NO), neuronal NOS (nNOS) and inducible NOS (iNOS) were detected by nitrate reduction method and colorimetric method. The expressions of nNOS and iNOS after reperfusion were analyzed by immunohistochemistry and Western blot assay. Results (1) The neu-rological scores after cerebral ischemia were significantly lower in Rg1-L group, Rg1-M group and Rg1-H group than those of cerebral ischemia-reperfusion group(2.40±0.55,1.80±0.84, 1.60±0.89 vs 3.20±0.84,P<0.05). (2) Compared with those of model group, serum levels of NO and iNOS were reduced, and nNOS levels increased, in three groups of Rg1. (3) Compared with those of model group, the expression of nNOS was significantly increased,and iNOS expression was significantly re-duced, in three groups of Rg1. Conclusion The preventive effects of Ginsenosides Rg1 on cerebral ischemia-reperfusion injury may be associated with the activation of nNOS and the inhibition of iNOS.

BACKGROUND:In vitro experiment has shown that the survival time of conventional chemical induction-induced neuron-like cells differentiated from bone marrow mesenchymal stem cells(BMSCs)was short,which limited its further application.With regard to the possibility of extension of iodine-induced neuron-like cells,the survival time has not yet been professionally reported.OBJECTIVE:To research the effects of the micro-element iodine on the survival time of neuron-like cells differentiated by BMSCs.METHODS:Rat mesenchymal stem cells at passage 3 were obtained under sterile condition,and divided into groups A-F.In group A,iodine ion was not added.In groups B-F,iodine ion at mass concentrations of 2,55,90,125 and 2 500 mg/L was added respectively.An additional blank control group was established,and simultaneously the cells were induced into neuron-like cells with dimethyl sulphoxide(DMSO).Cells following induction were subjected to immunohistochemistry.Survival time of neuron-like cells was observed under different mass concentrations of iodine ion.RESULTS AND CONCLUSION:When mass concentrations of iodine ion were between 55-125 mg/L,the survival time of neuron-like cells prolonged to about 5 days and structures of induced cells were intact.From then on,the number of dead cells was gradually increased till approximately one week,all neuron-like cells died.When mass concentrations of iodine ion were 2 mg/L and 2 500 mg/L,cell survival time was from 12-36 hours.No significant difference was determined compared with group A.Till 2 or 3 days,all neuron-like cells died.Above-described results indicated that an appropriated concentration of iodine iron added in the common chemical induction may be benefit for the survival time of the neuron-like cells differentiated by BMSCs,but the effect may be negligible for the survival time of neuron-like cells induced when the added concentration of iodine iron is too low or too high.

BACKGROUND:With increased age,bone marrow mesenchymal stem cells(BMSCs)are influenced with regard to quantity and quality,which will induce great damages to the donors.Many studies have focused on seeking substitute MSC source.In contrast it remains controversial whether umbilical cord blood contains MSCs.OBJECTIVE:To isolate MSCs from human umbilical cord blood,and to detect their biological properties.METHODS:Umbilical cord blood samples were sterilely isolated using Percoll density gradient centrifugation to harvest intermediate layer cells.DMEM medium containing fetal bovine serum,penicillin,streptomycin and L-glutamine was added.Following several adherences and purification,the floating cells were discarded.Thus,many adherent cells with a confluence were collected.When cells were 60% 80% confluent,cells were digested by trypsin for subculture.Cells at passages,1,5 and 9 were obtained and their morphological changes were observed.Cell surface antigens were measured using flow cytometry.Growth curves were drawn,and cell viability was determined utilizing MTT.RESULTS AND CONCLUSION:Isolated umbilical cord blood MSCs presented an even size,showing spindle or star-shape fibroblasts-like cells.Umbilical cord blood MSCs at 1,5,9 passages were greatly positive for CD29,CD105 and CD166,but weakly positive for CD34 and CD45.Following 5 days of incubation,cells entered logarithmic growth phase.The number was decreased at day 9.Population doubling time was(53.5±8.32)hours.Cells grew well.Cells at 1-7 passages showed similar viability(P ＞ 0.05).Till passage 9,cell proliferation viability was decreased,but no significant difference was determined(P ＞0.05).Results verified that MSCs can be successfully isolated from umbilical cord blood in vitro.Cells at passages 1-9 presented a good reproductive activity.

BACKGROUND:Wnt signaling pathway is the key to regulation of cell proliferation and differentiation.The evidence suggests that this pathway participates in the neural precursor cell proliferation,differentiation and determination of the regulation of cell fate.At present,the Wnt signaling pathway effects on the mesenchymal stem cells(MSCs)differentiated into neuron-like cells have not been reported.OBJECTIVE:To seek Wnt signal molecule that promotes the differentiation of MSCs into neuron-like cells.METHODS:MSCs were isolated from the femur of Sprague Dawley rats in vitro using the density gradient centrifugation,and then cultured.Following passage,cell surface markers CD29,CD44,CD34 and CD45 were measured using morphology and flow cytometry.These cells were selected and evaluated.Using neurotrophic factor and basic fibroblast growth factor combined with Wnt3a and Wnt5a induction scheme,effects of Wnt3a and Wnt5a during the differentiation of MSCs into neuron-like cells were compared utilizing immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR).Simple culture of basic fibroblast growth factor served as controls.RESULTS AND CONCLUSION:Following culture and passage of MSCs,cells adhered to the wall,showing even spindle shape.Flow cytometry showed great expression of CD29 and CD44 and low expression of CD34 and CD45.Following Wnt3a induction,cells were positive for nestin,neuron specific enolase,but no significantly expressed glial fibrillary acidic protein.Following induction,cell viability was good.In the Wnt5a induction and control groups,weakly positive expression of nestin was found,but neuron specific enolase and glial fibrillary acidic protein were negative.RT-PCR results demonstrated that nestin expression was detected in the Wnt3a induction group before and after induction.Significant amplified bands for neuron specific enolase were detected at day 5 following induction,and became more significant at day 10.Weak bands for glial fibrillary acidic protein were visible at day 10 following induction.These indicated that Wnt3a can promote the differentiation of MSCs into neuron-like cells in vitro.

Objective To investigate the effects of zinc fingers and homeoboxes 3 (ZHX3) silence on expressions of smad3, smad4 and RUNX2 in bone marrow mesenchymal stem cells (BMSCs). Methods ZHX3 low expression vector (ZHX3 silent group) was constructed and was transfected to rat BMSCs. Empty vector was transfected into BMSCs and was used as vehicle control group, and wild type BMSCs was used as the control group. The cell transfection rate was measured under a fluorescence microscope, and then the successful transfection was identified. The immunocytochemistry and immu?noblotting methods were used to detect the expression levels of smad3, smad4 and RUNX2. Results (1) Cells with BMSCs phenotype can be obtained by recovery culturing. (2) After transfection, the green fluorescent protein was found in ZHX3 si?lence group and vehicle control group. Blank control group showed no significant fluorescence. The expression level of ZHX 3 was significantly lower in ZHX3 silence group than that of vehicle control group. (3) Results of immunofluorescence asssay showed that the positive expressions of smad3 and smad4 were located in nucleus and cytoplasm, the positive expression of RUNX2 was mainly located in nucleus. Positive cells were observed in three groups. There was no significant difference in fluorescence intensity between the control group and the vehicle control group, but the fluorescence intensity was significant?ly lower in ZHX3 gene silence group than that of two control groups. (4) There were no significant differences in expressions of smad3, smad4 and RUNX2 betweem control group and the vehicle control group, but they were significantly higher than those of ZHX3 silence group(P < 0.05). Conclusion ZHX3 gene silence can delay vitro osteogenesis of BMSCs, which may play a role by the down-regulated expression levels of smad3, smad4 and RUNX2.