OBJECTIVETo observe the effect of Guizhi plus Gegen Decoction (GGD) on ultrastructural changes of intervertebral disc annulus fibrosus cells.

METHODSRats' intervertebral disc annulus fibrosus cells were isolated and cultured using adherence wall screening method. After annulus fibrosus cells were intervened by GGD, the microstructure and ultrastructural features of untreated annulus fibrosus cells and annulus fibrosus cells treated by GGD containing serum at different concentrations were observed under optical microscope and electron microscope.

RESULTSUnder optical microscope, most annulus fibrosus cells showed irregular polygons and few in star shape with rich superficial ecphyma. The nuclei were oval, large and complete. Under electron microscope, most cells in the blank group were oval after intervened by GGD containing serum at different concentrations. The nucleus was large, deviated, and irregular, the heterochromatin scattered diffusely, partial mitochondria vacuolized, and rough endoplasmic reticulum dilated. In the low dose GGD group, increased mitochondria and condensed density could be seen. The rough endoplasmic reticulum were expanded, lipid drops or glycogen could be occasionally seen. In the middle dose GGD group, increased endoplasmic reticulum expansion and condensed density could be seen. More medium density protein sediment could be seen. Increased mitochondria with condensed density could be seen, showing irregular cystic form with various sizes nucleus. In the high dose GGD group, increased rough endoplasmic reticulum with obvious expansion could be seen. More high density protein sediment could be seen. The nuclei were deviated. More mitochondria could be seen with secretory granules in them.

CONCLUSIONSAfter intervened by GGD containing serum at different concentrations, the ultrastructure of annulus fibrosus cells were manifested as follows: (1) The endoplasmic reticulum increased more in the middle and high dose GGD groups than in the blank group and the low dose GGD group. Greater density protein sediment occurred, especially in the high dose GGD group. (2) GGD played an important role in preventing ultrastructural changes induced by the degeneration of annulus fibrosus cells.

Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Intervertebral Disc ; cytology ; drug effects ; ultrastructure ; Rats ; Serum

Objective To investigate the possibility and mechanism of Dangguibuxue Decoction in early prevention of the development of atherosclerosis (AS) based on the study of the effect of Dangguibuxue Decoction on the signal transduction iter of nuclear transcription factor-kappa Bp65 (NF-κBp65) in monocyte activated by oxidized low-density lipoprotein (ox-LDL) in RAW264.7 cells. Methods The rabbits were randomly divided into the control group and medicated serum group and then given respectively normal saline and Dangguibuxue Decoction orally for preparing the medicated serum. The suspension of RAW264.7 cells was vaccinated into culture flask and cultured for 24 hours, and then divided into the blank control group, ox-LDL group, inhibitor group and medicated serum group. Except the blank control group all other groups were given 100 mg/L ox-LDL to activate RAW264.7 cells. After incubation the cells were collected. The expression of NF-κBp65 was detected by using immunohistochemistry assay, and the quantity of activated protein in RAW264.7 cells was detected by using immunoblotting technique. Results In ox-LDL group there were a lot of brown-yellow positive granules observed in the cytochylema and nucleus of RAW264.7 cells, and less in the medicated serum group. The ratio of protein band gray scale was 1.288 3±0.013 9 in the ox-LDL group and 0.918 9±0.015 3 in the medicated serum group. There was a significant difference statistically between two groups (P<0.05). Conclusion Dangguibuxue Decoction can down-regulate the protein expression and activation of NF-κBp65 in RAW264.7 cells stimulated by ox-LDL. It may be one of the mechanisms of Dangguibuxue Decoction for antagonizing AS that the signal transduction iter of NF-κBp65 was blocked.

Objective To investigate the mechanism of Dangguibuxue Decoction in anti-atherosclerosis (AS).Methods The medicated serum of Dangguibuxue Decoction was prepared. The cell activity was detected by using trypanblue staining. The protein activities of tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were detected by applying Western blotting method.Results After RAW264.7 cells were cultured for 3 days the cell activity with trypanblue staining was higher than 95 %. The result of Western blotting method showed that protein activity of TNF-α was promoted significantly after RAW264.7 cells were stimulated by oxidized low-density lipoprotein (ox-LDL). The comparison between the Dangguibuxue Decoction serum group and ox-LDL group showed a difference (P<0.05). The protein activity of ICAM-1 was promoted significantly after RAW264.7 cells were stimulated by ox-LDL. The comparison between the Dangguibuxue Decoction serum group and ox-LDL group showed a difference (P<0.05).Conclusion The medicated serum of Dangguibuxue Decoction can reduce the protein expressions of TNF-α and ICAM-1 to a certain degree, which may be one of the mechanisms of Dangguibuxue Decoction in anty-AS.

To compare the growth characteristics of non-hematopoietic adult stem cells (NASC) derived from rat fetal blood and rat bone marrow in vitro, and to study the differentiation of these stem cells into neuron-like cells in vitro, the fetal blood of pregnant rats and bone marrow of adult rats were sterilely collected; mononuclear cells (MNC) were isolated by using standard Ficoll-hypague techniques and then cultured in DMEM/LG containing 10% fetal bovine serum (FBS). The acquired NASCs were subcultured for passage. The immunophenotype of NASCs was detected by flow cytometry. The expanded NASCs were induced to differentiate into neurons-like cells by beta-mercaptoethanol (beta-ME), dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA). The specific markers of these neuron-like cells were detected by immunocytochemistry. The results showed that two kinds of subcultured NASCs showed homogeneous spindle-shaped and expressed antigens CD44 and CD54, but did not expressed CD11b and CD45. The both induced cells were similar to neuron in morphology and were positive for nestin and neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). It is concluded that no significant difference of NASCs derived from pregnant rat fetal blood and adult rat bone marrow found in cell morphology and biological characteristics. NASCs of both origins can be induced to differentiate into neuron-like cells, so fetal blood can be regarded as another resource of NASC.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; physiology ; Female ; Fetal Blood ; cytology ; Male ; Multipotent Stem Cells ; cytology ; physiology ; Neurons ; cytology ; Pregnancy ; Rats ; Rats, Wistar ; Stem Cells ; cytology

OBJECTIVECord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.

METHODSCB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.

RESULTSDifferent results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.

CONCLUSIONEx vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.

Animals ; Antigens, CD34 ; Cell Culture Techniques ; methods ; Cells, Cultured ; Culture Media ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Megakaryocytes ; cytology ; Mice ; Mice, SCID

OBJECTIVETo evaluate results of 1310 knees of meniscal treatments by arthroscopy and to present good method of meniscal treatment.

METHODSTheir age, traumatic mechanism and effects under arthroscopy were analysed in a series of 254 patients with meniscal injuries, there were 880 males and 374 females, the average of the patients was 25.5 years. The meniscal repair in 68 knee, partial meniscectomy in 756 knee, total meniscectomy in 480 knee and conservative treatment in 6 cases.

RESULTSThe patients were followed up 1 to 12 years with the average of 4 years and 3 months. The mean Lysholm score was 86.3 points in arthroscopic meniscal repair, 84.0 points in partial meniscectomy and 76.1 points in total meniscectomy. The mean Lysholm 98.7 points in all children patients with conservative treatment. They showed significant difference (P < 0.01) in the results of three treatments.

CONCLUSIONSMeniscal injuries should not cut off as it, should be repaired in 5 mm from meniscus to synovium and or partial meniscectomy. The general adoption is not the surgical operation on meniscal injuries of the child.

Adolescent ; Adult ; Age Factors ; Arthroscopy ; Casts, Surgical ; Child ; Female ; Follow-Up Studies ; Fracture Fixation ; methods ; Humans ; Knee Injuries ; surgery ; Male ; Menisci, Tibial ; surgery ; Middle Aged ; Tibial Meniscus Injuries ; Treatment Outcome

BACKGROUNDAdipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms.

METHODSFully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0-50 μg/ml) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber.

RESULTSOxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 μg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPβ protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPβ protein level in oxLDL-induced 3T3-L1 adipocytes.

CONCLUSIONSOxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

3T3-L1 Cells ; Animals ; CCAAT-Enhancer-Binding Protein-beta ; analysis ; physiology ; Chemokine CCL2 ; genetics ; secretion ; Cyclic AMP ; physiology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; Humans ; Lipoproteins, LDL ; antagonists & inhibitors ; pharmacology ; Mice ; Peptides ; pharmacology ; Signal Transduction ; physiology

OBJECTIVETo discuss the reasons for the operation performed on 13 patients with upper cervical disease and to explore the management and prevention of upper cervical disease.

METHODSThirteen patients with upper cervical disease were retrospectively reviewed. The reason for of reoperations on these patients were analyzed. The measures to reduce upper cervical operational complication and bad prognosis were discussed to avoid reoperations.

RESULTSThe reasons for reoperations included 9 cases with unstable or re-dislocated atlantoaxial joint, 10 cases with residual spinal cord compression, 1 case with malposition of odontoid screw, 1 case with adjacent cervical spine regression, 1 case with occipital-cervical fusion failure, 1 case with spinal cord injury during operation, 1 case with bone-plant slipped into canales spinalis, and 1 case with demand to take out internal fixation for aggravated symptom.

CONCLUSIONSThe common reasons for upper cervical reoperations were due to instability or redislocation of atlantoaxial joint and residual of spinal cord compression. Some measures such as reducing operate miss, using firm internal fixation and decompressing were advisable to decrease the incidence of reoperations.

Adolescent ; Adult ; Atlanto-Axial Joint ; Cervical Vertebrae ; surgery ; Decompression, Surgical ; Female ; Humans ; Joint Instability ; etiology ; prevention & control ; surgery ; Male ; Middle Aged ; Postoperative Complications ; etiology ; prevention & control ; surgery ; Reoperation ; statistics & numerical data ; Spinal Cord Compression ; etiology ; prevention & control ; surgery ; Spinal Fusion ; Young Adult

OBJECTIVEMesenchymal stem cells (MSCs) from bone marrow are capable of differentiating into cells of different tissue lineages such as bone, cartilage, and adipose tissue and are the best candidates for tissue engineering. It is well accepted that umbilical cord blood (UCB) is a source for hematopoietic stem cells. However, controversy exists as to whether UCB contains MSCs and can serve as a source of MSCs. Therefore, the aim of this study was to explore the biological characteristics and inducing differentiation ability of in vitro expanded UCB MSCs.

METHODSUCB was collected on normal full term delivery of infants with informed consent (n = 35) obtained from the mothers. Mononuclear cells (MNCs) were isolated from UCB by gravity centrifugation and cultured with DMEM including 10% fetal bovine serum. The morphology was observed under microscope per day. Cytochemical staining was carried out and flow cytometry was used to examine the surface antigen phenotype. Fifth passage cells were transferred into a different medium and osteogenic differentiation, adipogenic differentiation, and neurogenic differentiation were assessed.

RESULTSMSCs could be isolated and cultured from MNCs of a few UCB sources. These cells displayed fibroblast-like morphology. They withstood over 20 passages without significant structural changes. These MSCs were negative for alkaline phosphatase (ALP) staining and positive for alpha-naphthol butyric acid esterase (NBE) staining. Expression of CD(29), CD(44)and CD(105), especially the human MSCs-specific markers SH-2 and SH-3 were observed, but CD(3), CD(14), CD(19), CD(34) and CD(45) could not be found, indicating that these cells were not of hematopoietic origin. Exposure of these MSCs to serum-free osteogenic condition, they could differentiate into bone cells and form mineralized matrix as evidenced by Alizarin red staining 2 weeks later. When these UCB-derived MSCs were cultured in adipogenic medium, morphologic changes in cells as well as the formation of neutral lipid vacuoles were noticeable as early as 1 week after induction and visualized by staining with oil-red O. Surprisingly, these MSCs were also able to differentiate into neuroglial-like cells. Morphology of these induced cells resembled that of neurons. Immunocytochemistry showed that they expressed Nestin and neuron-specific enolase (NSE), but not glial fibrillary acidic protein (GFAP).

CONCLUSIONUCB does contain MSCs. These MSCs, which are multipotent, could be isolated and cultured from a few UCB sources. UCB might serve as an alternative source of MSCs to bone marrow.

Alkaline Phosphatase ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Centrifugation, Density Gradient ; Culture Media, Conditioned ; Fetal Blood ; cytology ; Flow Cytometry ; Histocytochemistry ; Humans ; Infant, Newborn ; Mesenchymal Stromal Cells ; metabolism ; Phosphopyruvate Hydratase ; metabolism

Objective To explore the applicative effect of the case-oriented and problem-targeted nursing teaching grand rounds in the department of cardiovascular. Methods A total of 57 nurse students were divided into two groups, 29 bachelor practice nurse students in the department of cardiovascular from July 2008 to July 2009 as research group (n = 29 ) and 28 bachelor practice nurse students in the department of cardiovascular from July, 2007 to July, 2008 as control group ( n = 28). The research group adopted the caseoriented and problem-targeted nursing teaching grand rounds mode, while the control group adopted the traditional nursing teaching grand rounds mode. Results The satisfied rate of the research group was higher than that of control group(95.9% vs 88% , P ＜0.05). The students' scores of the assessment exam after the intern in the research group was better than the students' in the control group( P ＜ 0.05 ). Conclusions There was a significant effect in the adopting of the case-oriented and problem-targeted nursing teaching grand rounds mode.This mode can improve the overall quality and clinical practice skills of nursing students, stimulate the motivation of self-learning, expand the visual thinking, promote the alteration of the teacher' s role and enhance the teacher's overall comprehensive capacity.