OBJECTIVE To study the immunosuppressive effect of combined therapy with FK506 and RS-61443 in rat limb allotransplantation. METHODS: A total of 101 male SD rats were randomly divided into seven groups and used as recipients, and 101 Wistar rats were used as donors. All SD rats were performed limb allotransplantation without using immunosuppressants in control group. In experimental groups (Groups 1-6), the recipients were immunosuppressed with various dosages of FK506, RS-61443 or FK506 + RS61443, after transplantation for 5 weeks. To evaluate the results, we observed circulation of the transplanted limb, the mean rejection time, the histologic grading of skin rejection of limb grafts and the survival time of limb grafts. RESULTS: The control group showed rejection signs (edema and erythema of the skin) after a mean time of 3.36 +/- 1.15 days, and the mean survival time of the allografts was only 7.00 +/- 0.78 days. In the groups only using FK506 or RS-61443, the survival time were prolonged to varying degrees, but rejection occurred even in the period of using drug. As dosage increased, the rejection could not be prevented and the damage to liver and kidney could be induced. In the group using FK506 in combination with RS-61443, only skin and muscle of limb allografts showed slight rejection sign, function of liver and kidney was not obviously affected, the mean survival time of limb allografts was prolonged to 58.76 +/- 6.81 days. CONCLUSIONS: A combination of FK506 and RS-61443 is a more potent immunosuppressive agent than FK506 oro RS-61443 in preventing the rejection of limb allografts, and it can obviously prolong the survival time of limb allografts.
Objective To determine the contents of matrix metalloproteinase 3 (MMP-3) and interleukin 1 (IL-1) in the tissues of the lumbar disc herniation and to investigate their roles in the pathogenesis. Methods The tissues of the herniated lumbar disc were obtained from 30 patients undergoing surgery for persistent radiculopathy from June 2003 to December 2004 and at the same time these samples were divided into the following three experimentalgroups: the bulge group (n=11), the protrusion group (n=9), and the prolapsus group (n=10)，14 males, 16 females, aged 33.64 years. As the control group, 9 lumbar disc specimens were harvested from 9 patients(4 males, 5 females, aged 21-58 years) suffering from bursting fracture of the lumbar spine. The specimens were analyzed by the ELISA method for the contents of MMP-3 and IL-1. Results The contents of MMP-3(14.25±1.32, 19.89±2.97,20.69±2.18 ng/ml in the bulge group, protrusion group and prolapsus group, separately) and IL-1(8.52±0.22， 11.88±0.52，11.90±0.73 pg/ml in the bulge group, protrusion group and prolapsus group, separately) in the experimental groups were significantly higher than those in the control group. The contents of MMP-3 and IL-1 in the protrusion group were not significantly higher than those in the prolapsus group, but they were significantly higher than those in the bulge group(P＜0.01). The contents of MMP-3 had a significant relationship with the contents of IL-1 in the three experimental groups and the control group(P＜0.01). Conclusion The result demonstrates that the tissues of the lumbar disc herniation can produce both MMP-3 and IL-1, which may have an unknown but important relationship with each other.
OBJECTIVE: To investigate the effects of three-dimentional culture in bioderived material modified by Pluronic F-127 on the growth and function of rabbit periosteal osteoblast in vitro. METHODS: Bio-derived materials were from fresh pig ribs and were modified by Pluronic F-127. Then rabbit periosteal osteoblasts were cultured in bio-derived materials(group A), in the modified bio-derived materials(group B) and on the plastic surfaces as a control (group C), respectively. During a 7-day period, the status of growth, cell viability and alkaline phosphatase(ALP) activity were measured. RESULTS: Osteoblasts attached, elongated and grew well on the modified bio-derived materials. There were no significant difference in osteogenesis and ALP activity between group A and group B(P gt; 0.05). The osteogenesis and ALP activity in groups A, B were less than those in group C (P lt; 0.01). CONCLUSION: Pluronic F-127 can be used for a carrier for bioactive factors to modify bio-derived material.
Objective To investigate the heterotopic odontogenesis ability ofDelta1 gene transfected human dental pulp stem cell (DPSC) and nanohydroxyapatite/collagen (nHAC) composite scaffold. Methods The cultured human DPSC was transfected with Delta1-enhanced green fluorescent protein recombinant retrovirus supernatant，and was selected by puromycin to obtain the positive cell clone. The experimental group contained the Delta1 transfected DPSC; however, the control group did not contain the Delta1 transfected DPSC but contained DPSC transfected with vectors only. The cells were seeded into the nHAC carriers and were cultured in the odonto-inductive medium. The growth of the transduced cells in the carriers was observed by the fluorescent phase contrast microscope and the scanning electron microscope (SEM). The cell-carrier composites were subcutaneously transplanted into the Delta1 transfected 8 nude mice (female, 8 weeks old). Eight weeks after operation,the composites were taken out and tested with the histological and the immunohistological methods.Results Green fluorescence was observed inthe cells in the experimental group, which were grown in the carriers by the fluorescent phase contrast microscope. Observed by SEM, great amounts of transduced DPSC were observed along the scaffold materials, even filling the porous structures of nHAC and secreting a lot of extracellular matrix. However, in the control group, much fewer cells were found in the carriers. All the 4 Delta1 transduced DPSC-nHAC composites produced dentin-like structures that lined the surfaces of some nHAC porous structures. The odontoblast-like cells extended the cytoplasmic processes into the dentinal matrix, which was interfaced with a pulp-like interstitial tissue infiltrated with the blood vessels. Dentin sialophosphoprotein was expressed in the odontoblast-like cells when immunohisochemistry was performed. The morphology of the control composite was a typical one of the fibrous connective tissue,and only a little dentin-like structure was found in 2 of the 8 control transplants. Conclution DPSC can be used as the recipient cell of the Delta1 gene for expression and secretion of the Delta1 protein. The composites of the transfected cells and nHAC can induce heterotopic odontogenesis, which indicates that Delta1 is a novel candidate for the gene enhanced dentinpulp composite engineering.
OBJECTIVE: To observe the changes of heme oxygenase-1 (HO-1) expression in the skeletal muscle after ischemia-reperfusion of hind limb in rats. METHODS: A model of hind limb ischemia was made by clamping femoral artery with a microvascular clip. Soleus muscle was obtained from the animals received sham operation, 4 h ischemia without reperfusion and 2 h, 4 h, 8 h, 16 h, 24 h reperfusion after 4 h ischemia. Soleus histology and malondialdehyde (MDA) content were measured. The levels of HO-1 mRNA and protein were measured in different time by Northern blotting, Western blotting and immunohistochemistry technique. RESULTS: After ischemia-reperfusion of limb, HO-1 mRNA increased at the 2nd hour, reached a peak at the 8th hour, and returned toward baseline at the 24th hour. The change of protein level was essentially in agreement with that of mRNA. Immunohistochemical results showed that HO-1 expressed primarily in skeletal muscle cytoplasma. There were no positive signals of mRNA and protein in sham group and in ischemia group. After limb reperfusion, MDA contents in the soleus muscle increased significantly when compared with that in the sham group (P lt; 0.05). MDA content of the 8th after reperfusion decreased significantly when compared with that of the 4 h after reperfusion (P lt; 0.05). CONCLUSION: Ischemia-reperfusion can induce HO-1 expression in skeletal muscle in rats, which may provide protection for injured tissue.
ObjectiveTo observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV).MethodsForty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV.ResultsThe results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P＜0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000).ConclusionsThe expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.
【Abstract】Objective Stromal cell-derived factor-1(SDF-1, CXCL12) is a member of the CXC subfamily of chemokines which, through its cognate receptor (CXCR4), plays an important role in tumor invasion and metastasis. This study analyzed quantitatively the expression of SDF-1 and its relation with clinicopathologic feature and clinical outcome in human breast cancer.Methods Expression of SDF-1 mRNA in 8 breast cancer cell lines, an endothelial cell line HECV and a fibroblast cell MRC5 was studied by using RT-PCR. In addition, the expression of SDF-1 was investigated at both protein (immunohistochemistry) and mRNA(real-time PCR) levels in a group of human normal mammary(n=32) and tumour tissues(n=120). Results SDF-1 expression was identified in MRC5, MDA-MB435s, MDA-MB436, MCF7 cell lines, breast tumour and normal tissues. Significantly higher level of SDF-1 was seen in lymph node positive than in lymph node negative tumours (399.00±210.00 vs 0.89±0.47), P=0.048. The level of SDF-1 expression in patients who developed local recurrence or metastasis, or patients who died of breast cancer was higher than in patients who were disease free as well, (670.00±346.00 vs 0.83±0.35), P=0.01. It was most notable that level of SDF-1 was significantly correlated with over survival (P=0.01) and incidence free survival (P=0.035, by Cox proportion analysis).Conclusion SDF-1 is a factor that is expressed in both stromal cells and some breast cancer cells. Its level are correlated with lymph node involvement, prognosis and survival in patients with breast cancer. SDF-1 may therefore have a potential prognostic value in breast cancer.
Objective To evaluate the effect of WO-1 on repair of the bone defect in the New Zealand rabbit radius by an oral or local administration. Methods Bone defects were surgically created in the bilateral radii of 36 Zealand rabbits (1.6-2.0 kg), which were randomly divided into3 groups. In Group A, the defective areas were given WO-1 0.1 ml (50 mg/ml) by the local injections; in Group B, the rabbits were given WO-1 5 mg each day by the oral administration. Group C was used as a control group. Among each of the 3 groups, 4 rabbits were randomly selected and were sacrificed at 20, 30 and 60 days after operation, respectively. Then, the serological, X-ray and histological examinations were performed. Results The serum alkaline phosphatase and bone glaprotein levels were significantly higher at 20 and 30 days after operation in Groups A and B than in Group C, but significantly lower at 60 days after operation in Groups A and B than in Group C（Plt;0.01）. The X-ray and histological examinations at 20, 30 and 60 days after operation revealed that the callus formation and remodeling were earlier in Groups A and B thanin Group C, and the remodeling was earlier and better in Group A than in Group B. Conclusion WO-1 can promote the repair of the radial defect in a rabbit; however, further studies on the doseeffect relationship, administration time, and administration route are still needed.
Objective To explore the expression of CD105 protein in esophageal squamous cell carcinoma and it＇s relationship with P53 protein. Methods Using streptavidin biotinperoxidase （SP） method, the expression of CD105 protein and P53 protein in esophageal squamous cell carcinoma were examined in normal esophageal tissues （n=10） and esophageal squamous cell carcinoma tissues（n=86）. Results The expression positive rate of CD105 protein was 74. 4%（64/86） in esophageal squamous cell carcinoma , 0% in normal esophageal epithelium. Expression positive rate of CD105 protein was 66. 1%（37/56） in early stage （stage Ⅰ-Ⅱ ）, 90.0% （27/30） in later stages （stage Ⅲ-Ⅳ ）. The expression of CD105 protein were bly associated with P53 protein（P〈0. 05）. Conclusion CD105 protein may participate in the onset and progression of esophageal squamous cell carcinoma. CD105 protein could he a new diagnostic /therapeutic target in esophageal squamous cell carcinoma.
Objective To investigate the serumlevel of endothelin-1 ( ET-1) in patients with acute lung injury/acute respiratory distress syndrome ( ALI/ARDS) and its clinical significance. Methods Thirty-one ALI/ARDS patients received mechanical ventilation in ICUand 25 normal subjects were recruited in the study. The patients who died in two weeks fell in death group, and the patients who did not died in two weeks fell in survival group. The serum level of ET-1 measured by EIA method were compared between thepatients with different severity of lung injury [ evaluated by American-European Consensus Conference on ARDS ( AECC) criteria and lung injury score( LIS) ] , and between the patients with different prognosis ( death or survival ) . The correlation was analyzed between the level of ET-1 and clinical parameters.Results The ET-1 level was higher in the ALI/ARDS patients than that in the control subjects [ ( 6. 18 ±4. 48) ng/L vs. ( 2. 68 ±1. 34) ng/L, P lt;0. 05] . There was no significant difference in the patients with different severity [ ALI vs. ARDS, ( 5. 43 ±4. 39) ng/L vs. ( 7. 01 ±4. 51) ng/L, P gt; 0. 05; LIS≤2. 5 vs.LISgt;2. 5, ( 5. 93 ±5. 21) ng/L vs. ( 6. 68 ±2. 76) ng/L, P gt; 0. 05] . The ET-1 level in the death group continued to increase, and higher than that in the survival group on the 5th day [ ( 7. 96 ±3. 30) ng/L vs.( 4. 36 ±3. 29) ng/L, P lt; 0. 05] . The ET-1 level was positively correlated with SIRS, SAPSⅡ and APACHEⅡ ( r = 0. 359, 0. 369 and 0. 426, respectively, P lt; 0. 05 ) , and negatively correlated with PaO2 /FiO2 and AaDO2 ( r = - 0. 286 and - 0. 300, respectively, P lt;0. 05) . Conclusion The measurementof serum ET-1 can help to evaluate the severity and prognosis of ALI/ARDS patients.