Objective To observe the effects of cryopreservation and resuscitation on the biological characteristics of mesenchymal stem cells (MSCs) derived f rom human umbilical cord blood. Methods MSCs were isolated and cultured f rom human umbilical cord blood in vitro. The cells were passaged , and the third generation of MSCs were cryopreserved in－196 ℃ liquid nitrogen for 4 weeks with cryopreservation medium , which contained 10 % dimethyl sulfoxide (DMSO) and 90 % fetal calf serum ( FCS) . The morphology , proliferation and differentiation of MSCs were investigated and compared with those of MSCs before cryopreservation. Results There was no significant difference of morphology between pre-cryopreserved MSCs and the ones af ter resuscitation. It was observed that all MSCs were spindle-shaped and showed adherence growth characteristic before and af ter cryopreservation. The cell growth curves of MSCs were also similar before and af ter cryopreservation. Even though the curve of resuscitated MSCs descended a little as compared with that of pre-cryopreserved MSCs , there was no significant difference ( Pgt; 0. 05) . After 2-week adipocytic differentiation induction , fat drops could be found in the kytoplasm of MSCs and they were red when stained with oil-red O staining , which suggested that MSCs could be induced and differentiated into adipocytes. Af ter 4-week osteoblastic differentiation induction , MSCs could be induced and differentiated into osteoblasts , and calcium node showed black when stained with Von Kossa staining. There were no significant changes of the differentiating ability of MSCs into adipocyte and osteoblast before and after cryopreservation. Conclusion MSCs derived from human umbilical cord blood maintains their biological characteristics af ter cryopreservation and resuscitation.
Objective To investigate the clinicopathological characteristics of proximal gastric cancer (PGC). MethodsThe clinical course and pathologic feature of 118 PGC patients were analyzed, and compared with those of 310 distal gastric cancer (DGC) patients. ResultsThe incidence of PGC was lower than DGC, the percentage of Ⅲ,Ⅳ stages and undifferentiated type in the PGC group were significantly higher than in DGC. For the surgical procedure, patients in the PGC had significantly higher percentages of total gastrectomy and other organ resection than in DGC. The percentage of patients with positive margin and lymph node metastasis in PGC was also significantly higher than in DGC. Esophageal invasion and lymph node metastasis were much more in PGC. The 5year survival of patients with PGC was significantly lower than that with DGC. No significant differences were found between the two groups with respect to the mortality rates and complications. Conclusion The relatively poor prognosis associated with PGC is mainly from advanced cases and esophageal invasion. Early detection and treatment is the most important strategy to improve the survival of patients with PGC.
【Abstract】 Objective To observe the effects of Angelica dahurica extracts on the biological characteristics of human keratinocytes (KC) in vitro and to explore the possible mechanism in promoting wound healing. Methods HaCaT cells of passage 5 from KC were used during the experiment. Different concentrations (5 × 10-2, 5 × 10-3, 5 × 10-4, and 5 × 10-5 g/L) of Angelica dahurica extracts, which was obtained by 95% ethanol from Angelica dahurica raw material, were prepared by DMEM containing 0.25% fetal bovine serum (FBS). After the extracts at different concentrations were respectively used for KC culture for 5 days, the cell proliferation activities were detected by MTT, and DMEM containing 0.25% FBS served as the negative control. According to the cell proliferation activity, the optimal concentration was determined. KC was further treated with Angelica dahurica extracts of the optimal concentration (experimental group) or with DMEM containing 0.25% FBS (control group) for 48 hours. The cell cycle was tested by flow cytometry. Cyclin D1 and Caspase-3 mRNA levels were also detected by real-time fluorescent quantitative PCR technique. Results Angelica dahurica extracts at concentrations of 5 × 10-4, 5 × 10-3,and 5 × 10-2 g/L could significantly enhance KC proliferation, showing significant differences in absorbance (A) values compared with that of control group (P lt; 0.05) with an optimal concentration of 5 × 10-3 g/L. At this concentration, an increased percentage of S and G2/M phase cells and a decreased percentage of G0/G1 phase cells were detected, showing significant differences when compared with control group (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that the cyclin D1 and Caspase-3 mRNA levels of experimental group was significantly down-regulated, showing significant differences when compared with control group (P lt; 0.05). Conclusion Angelica dahurica extracts can promote the proliferation of KC, accelerate the cell cycle of KC by down-regulating mRNA expressions of cyclin D1, and inhibit apoptosis by down-regulating mRNA expressions of Caspase-3. These effects might enhance the process of wound healing by expediting the process of epithelization.
Objective To evaluate the tensile mechanical characteristics of decalcified cortical bone matrix with different thicknesses so as to provide an experimental basis for the scaffold of tissue engineering. Methods Decalcified cortical bone matrix was prepared from fresh bovine tibia with rapid decalcification techniques. Its physical characteristics including colour, texture, and so on, were observed. Then the decalcified rate was calculated. Decalcified cortical bone matrices were radially cut into sl ices with different thicknesses along longitudinal axis and divided into 4 groups: group A (100- 300 μm), group B (300-500 μm), group C (500-700 μm), and group D (700-1 000 μm). Then the sl ice specimens of each group were characterized with tensile test and histological examination. Results General observation showed that decalcified cortical bone matrix with hydrogen peroxide treatment was ivory white with good elasticity and flexibil ity. The decalcified rate was 97.6%. The tensile strength and elastic modulus of groups B, C, and D were significantly higher than those of roup A (P lt; 0.05); there was no significant difference among groups B, C, and D (P gt; 0.05). The stiffness in 4 groups increased gradually with the increasing thickness, it was significantly lower in group A than those in groups B, C, and D (P lt; 0.05), and in groups B and C than that in group D (P lt; 0.05). While there was no significant difference in ultimate strain within 4 groups (P gt; 0.05). Histologically, intact osteon was observed in every group, with an average maximum diameter of 182 μm (range, 102- 325 μm). Conclusion The mechanical properties of decalcified cortical bone matrix might depend on the integrity of the osteons. Sl ices with thickness of 300 μm or more could maintain similar mechanical properties when decalcified cortical bone matrix is used as a scaffold for tissue engineering.
Objective To evaluate the cl inical effect and the pathological characteristics of acellular allogeneic dermal matrix in repairing unstable burn scar. Methods From January 2007 to June 2008, 19 cases of unstable burn scars (24 parts) were treated, including 16 males (20 parts) and 3 females (4 parts) with a median age of 27 years (range, 3-58 years). Theinjury was caused by flame (14 cases, 18 parts), electricity (4 cases, 5 parts), and hot water (1 case, 1 part). The unstable burn scars located on hands (8 cases), forearms (2 cases), thighs (3 cases), legs (2 cases), feet (2 cases), chest (1 case), and abdomen (1 case). Scar formed for 3 months to 1 year. The area of defect varied from 7 cm × 5 cm to 22 cm × 15 cm after scar removal. Defects were covered with acellular allogeneic dermal matrix and autogenous spl it-thickness skin graft. At 6-18 months after operation, the pathological observations of the epidermis, the basal membrane, and structural components of the dermis were done. Results All wounds healed by first intention. Scar ulcer disappeared completely in 18 cases and the composite skin grafts all survived. Some bl isters occurred in 1 case and were cured after dressing changing. All patients were followed up 10 months to 2 years (18 months on average). The grafted-skin was excellent in the appearance, texture, and elasticity. The function recovered well. Only superficial scar was observed at skin donor sites. Pathological observation showed that the epidermis and the basal membrane of the skin grafts were similar to that of normal skin, and no significant difference was found in newly capillaries between them. Collagen fibers arranged regularly, and there were few inflammatory cells in the matrix. Conclusion Acellular allogeneic dermal matrix with autogenous spl it-thickness skin graft may effectivly repair the wound after removing the unstable burn scar, and its structure is similar to that of normal skin.
Objective To summarize the cl inical characteristics of 596 patients with fracture in Wenchuan earthquake and to investigate the therapeutic methods and effects of early treatment. Methods From May 12th 2008 to May 21st 2008, 596 patients with fracture caused by Wenchuan earthquake were treated, including 283 males and 313 females aged1.9-102 years (median 43 years). The time from injury to hospital ization varied from 12 minutes to 4 days. There were 132 cases of upper extremity fracture, 496 cases lower extremity fracture, 10 cases clavicular fracture, 16 cases scapular fracture, 23 cases pelvis fracture, and 59 cases spinal fracture. Among them, 183 cases were open fracture and 413 cases were closed fracture. And 214 cases had multiple fracture (35.9%) and 68 cases had crush injury in l imbs which scored (6.84 ± 2.48) points according to the mangled extremity severity score (MESS). Thirty-six cases were combined with neurovascular injury. The wound of the open fracture was contaminated at different degrees, but no gas gangrene was observed. Open fracture was treated with suturing or no suturing after debridement, open reduction and internal or external fixation. Closed fracture was fixed with spl ints, cast and traction. Forty-nine patients whom were highly suspected as osseous fascia compartment syndrome received incision decompression timely, and 34 patients whose MESS were above 7.0 points or suffering from crush injury of l ifethreatening systemic symptoms received amputation. Results Apart from 34 patients receiving amputation, 460 patients achieved functional reduction of fracture after manipulative reduction and 102 cases got satisfactory reduction after surgery. Postoperatively, 289 patients were transferred to other hospitals. Among the rest 307 patients, 34 with severe wound infection were healed after multiple debridement, anti-infection, and skin flap transplantation (16 cases healed by first intention and 18 cases healed by second intention), 42 cases with crush syndrome were treated with open decompression and amputation, and nodeep venous thrombosis of lower l imb, stress ulcer and death were observed after operation (29 cases healed by first intention and 13 cases healed by second intention). Conclusion By aiming at the features of fracture caused by earthquake, the prompt and professional treatment can achieve good therapeutic effects.
【Abstract】 Objective To explore an effective method to cultivate esophageal mucosa epithel ial cells (EMECs)of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Methods Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. Results The primary culture of canine EMECs arranged l ike slabstone. Immunohistochemical staining of CK-19 of the2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. E MECs werepolygon in shape and arranged l ike slabstone, and formed a single layer on the surface of SIS. The cells were contact ed closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, andshowed laminate arrangement. Conclusion With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6 %FBS is a simple and feasible method. SIS shows good biocompatibil ity and can be used as a good scaffold material in th e tissue engineered esophagus.
Objective To explore a method to isolate, culture and multiplicate the placentaderived mesenchymal stem cells (PMSCs) and the bone marrow-derived mesenchymal stem cells (BMSCs) of rabbit，and to compare their biological characteristics. Methods PMSCs were isolated from placenta of 1fetation rabbitby Percoll density gradient centrifuge and cultured in vitro. BMSCs were isolated from hindlimb bone marrow blood of 1 new born rabbit by direct plates culturemethod. The 3rd passage PMSCs and BMSCs were observed by inverted phase contrast microscope. The stem cell marker (CD44, CD105, CD34 and CD40L) were examined by immunohistochemistry. The 2nd passage PMSCs and BMSCs were co-cultured with biomaterials，(1.0-1.5)×106 cells in one biomaterial， and then observed by aematoxylinstaining after 5 days,and by SEM after 3 days and 8 days. Results PMSCs and BMSCs were both uniformly spondle-shaped in appearance and showed active proliferative capacity. The proliferative ability of PMSCs were quite b and declined with passages. After cultured 10 passages in vitro, its growthslowed. Both PMSCs and BMSCs expressed CD44 and CD105,but did not express CD34 and CD40L immunoreactivity. PMSCs and BMSCs poliferated and adhered to the surface of biomaterials, and cell formed clumps and network; the cells proliferation and the matrix were seen in the pore after 5 days of culture. The observation ofSEM showed that many cells adhered to the biomaterials with spindle-shape and polygon after 3 days; and that PMSCs and BMSCs grew,arranged in layers andsecreted many matrices; the reticular collagen formed arround cells after 8 days. Conclusion PMSCs and BMSCs have similar biological characteristics and PMSCs can be served as excellent seedingcells for tissue engineering.
Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokinesinduction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β1(TGF-β1,10 ng/ml), basic fibroblast growth factor(bFGF,25 ng/ml) addexamethasone (DEX,10-7mol/L) with 15%FBS in DMEM. Cartilage slices wereobtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as shortspindle shape, while the 5th MSCs showed longspindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positivestain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them（Pgt;0.05）, and there was significant difference between groups A and D（Plt;0.05）. No significant difference was noted in groups A,B and C on proliferation by MTT（Pgt;0.05）,except that of at the fourth day after transfection between groups A and C（Plt;0.05）. RT-PCR demonstrated that MSCs always had higher levelsof mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.
OBJECTIVE This paper was to study the biological characteristics of the transformed human embryonic tendon cells, the relation between cell growth and culture conditions, and to compare these features with that of human embryonic tendon cells. METHODS The pts A58H plasmid had successfully used to transform a tendon cell line from human embryo in our past work. The human embryonic tendon cells and the transformed human embryonic tendon cells were cultured in vitro. In different culture conditions, the growth curve were drawn respectively. Population dependence and proliferation capability of the cells were investigated through plate cloning test and soft agar culture. The collagen secreted by cells was identified by immunohistochemical method. RESULTS In routine culture condition, the growth properties of the human embryonic tendon cell and transformed cells were almost identical. The growth properties of the transformed cells were not changed when the cells were frozen storage. There were changes of growth characteristics of the transformed cells when the culture temperature was changed. The transformed cells could subcultured continually and permanently. The proliferation capability of the transformed cells were ber than that of the human embryonic tendon cells. Moreover, the growth of the transformed cells was serum-dependent, and the phenomenon of contact inhibition was observed. The transformed cells were not able to grow on soft agar culture. They had the capacity of secreting collagen type I. CONCLUSION The transformed human embryonic tendon cells could be subcultured continually and permanently, and their growth could be controlled by changing their culture conditions and they had no malignant tendency in biological characteristics. They could be taken as an ideal experimental material for tendon engineering.