ObjectiveTo study the changes of levels of α subunits of stimulatory (Gsα) and inhibitory guanine nucleotide binding protein (Giα) in newborn guinea pig (0 2 days old) myocardium undergoing global ischemic reperfusion, and influences on the changes by St.Thomas Ⅱ and cold blood cardioplegic solution.MethodsThirty newborn guinea pigs were randomly assigned to three groups. GroupⅠ ( n = 10): the newborn hearts suffered by hypothermic global ischemia; group Ⅱ( n =10): the newborn hearts arrested by St. Thomas Ⅱ , and group Ⅲ ( n = 10): the newborn hearts arrested by cold blood cardioplegic solution. Levels of Gsα and Giα were investigated with Western blot analysis.ResultsNo differences of levels of Gsα and Giα were found in three groups before ischemia ( P gt;0.05). The level of Gsα after ischemia was significantly decreased than before ischemia in groupⅠand group Ⅱ ( P lt; 0 01), whereas no pronounced changes in group Ⅲ ( P gt;0.05) were noted after ischemia. The level of Gsα in group Ⅲ was not significantly changed after reperfusion compared with before ischemia( P gt;0 05), and it was much higher than those in groupⅠand group Ⅱ ( P lt; 0 01). Level of Giα was found not markedly changed in group Ⅲ after reperfusion compared with that before ischemia, but was notable higher in groupⅠand group Ⅱ( P lt;0.01). ConclusionsSignificant decrease of level of Gsα, whereas marked increase of level of Giα are found in myocardium of newborn guinea pig undergoing hypothermic (20℃) ischemic reperfusion. No impact of St. Thomas Ⅱ on these changes is verified, but recovery to the level of Gsα and Giα before ischemia is achieved by cold blood cardioplegic solution after ischemia and reperfusion. Unbalance between Gsα and Giα is the one of the mechanisms of ischemic reperfusion injury for immature myocardium.
Objective To study the clinical significance of DNA content and expression of nm23-H1, C-erb B-2 and p53 oncoproteins in hepatocellular carcinoma. Methods DNA content was measured after DNA feulgen’s dyeing, the expression of nm23-H1, C-erb B-2 and p53 oncoproteins was detected immunohistochemically. Results The incidence of aneuploid DNA content was 50.0% (12/24) in ≤5 cm group, and was 82.1% (23/28) in ＞5cm group; aneuploid DNA content was related with liver metastasis and cancerous thrombosis. The expression of nm23-H1 in HCC with liver metastasis was higher than that without metastasis. The positive rate of p53 in HCC with canerous thrombosis was higher than that without cancerous thrombosis. The positive rates of nm23-H1 and p53 in HCC with aneuploid DNA content were higher than those with diploid DNA content. The positive rate of C-erb B-2 did not have significantly difference between groups. Postoperative survival rates were possibly related with DNA content as well as expression of nm23-H1, and p53 oncoproteins. Conclusion Aneuploid DNA conten, expression of nm23-H1 and p53 oncoproteins are closely related with the invasiveness of HCC, and have remarkably clinical significance.
Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.
Objective To investigate the inhibitory effects of RNA interference (RNAi) expression vector on the expression of survivin in pancreatic cancer cell PANC-1. Methods The protein and mRNA expressions of survivin were examined with immunofluorescence and RT-PCR. The survivin gene was cloned into the T-vector and sequenced. The RNAi expression vectors targeting survivin, named si-svv-1 and si-svv-2 respectively according to whether they harbored a mutation or no mutation, were constructed and transfected into PANC-1 cells with liposome. The expression of survivin mRNA was detected with RT-PCR. Apoptosis of PANC-1 cells was analyzed with DNA ladder and FACS. Results There was a high degree expression of survivin in PANC-1 cells. The expression of survivin was not inhibited by RNAi expression vectors si-svv-1, but inhibited about (72.43±8.04)% by si-svv-2 and the apoptosis rate of PANC-1 cells increased to （12.36±1.44）% after 72 h. Conclusion The RNAi expression vector can effectively inhibit the expression of survivin in pancreatic cancer cell PANC-1 cells and induce the apoptosis in PANC-1 cells.
OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
【 Abstract 】 Objective To investigate the effects of 250 ml/m3 carbon monoxide (CO) inhalation or intraperitoneal infusion on lipopolysaccharide (LPS) induced rat intestinal tract injury, and to detect the roles of p38 mitogen-activated protein kinase (MAPK) pathway during CO administration. Methods After received 5 mg/kg LPS or an equal volume of normal saline by intravenous injection, 108 male SD rats were randomly divided into 6 groups: control group, CO inhalation (250 ml/m3) group, CO intraperitoneal infusion (250 ml/m3 at a rate of 2 L/min) group, LPS (5 mg/kg) group, LPS (5 mg/kg)+CO inhalation (250 ml/m3) group and LPS (5 mg/kg)+CO intraperitoneal infusion (250 ml/m3 at a rate of 2 L/min) group. The animals were differently sacrificed at 1, 3 and 6 h for the observation, and the ileum tissues were homogenized for determination the levels of platelet activator factor (PAF), intercellular adhesion molecule-1 (ICAM-1) and interlukin-10 (IL-10) with enzyme-lined immunosorbent assay, the content of maleic dialdehyde (MDA) with thiobarbitric acid, the activity of myeloperoxidase (MPO) with chemical method, the activity of superoxide dismutase (SOD) with hydroxylamine, the activity of phosphorylated p38 MAPK with Western blot, the pathology with light microscope, and the extents of cell apoptosis were showed by the ratio of the apoptotic cells which had less DNA to the total cells of a cell-suspension sample by using the flow cytometry after being stained with propidium iodide. Results Compared with both control, CO inhalation and intraperitoneal infusion group at the same time point, the levels of PAF, ICAM-1, MDA, MPO, cell apoptosis rate and the phosphorylated p38 MAPK protein in LPS group were increased, while IL-10 and SOD were decreased (P ＜ 0.05 or 0.01), and accompanied by severe intestinal tract injury. There were no statistics differences at the different time point in the same group. PAF, ICAM-1, MDA, MPO and cell apoptosis rate in both LPS+CO inhalation group and LPS+CO intraperitoneal infusion group were lower, while IL-10 and SOD were higher than the corresponding value in LPS group at the same time point (all P ＜ 0.05), with ameliorate injury too, but the expression of phosphorylated p38 MAPK was further up-regulated than that of LPS group (all P ＜ 0.05). However, there were no significant differences in these parameters between LPS+CO inhalation group and LPS+CO intraperitoneal infusion group. Conclusion 250 ml/m3 CO inhalation and intraperitoneal infusion exerts the similar protection against LPS induced rat intestinal tract injury via anti-oxidant, anti-inflammation, and anti-apoptosis. This may involve the p38 MAPK pathway.
Objective To study the adenovirus-mediated human bone morphogenetic protein-2 gene （Ad-hBMP-2）transferred to the intervertebral disc cells of the New Zealand rabbit in vitro. Methods The cells of New Zealand white rabbitswere isolated from their lumbar discs. The cells were grown in the monolayer and treated with an adenovirus encoding the LacZ gene (Ad-LacZ) and Ad-hBMP-2 (50,100, 150 MOI,multiplicity of infection) in the Dulbecco’s Modified Eagle Medium and the Ham’s F-12 Medium in vitro. Three days after the Ad-hBMP-2 treatment,the expression of hBMP-2 in the cells that had been infected by different dosesof MOI was determined by immunofluorescence and the Western blot analysis, and the expression was determined in the cells with the Ad-LacZ treatment in a dose of 150 MOI. Six days after the Ad-hBMP-2 treatment, mRNA was extracted for the reverse transcription polymerase chain reaction (RT-PCR) and the difference was detected between the control group and the culture group that was treated withAd-hBMP-2 in doses of 50, 100 and 150 MOI so that the expressions of aggrecan and collagen ⅡmRNA could be observed. Results The expression of hBMP-2 in the cells was gradually increased after the transfection in an increasing dose, which was observed by immunofluorescence and the Western blot analysis. At 6 days the aggrecan and collagen type Ⅱ mRNA expressions were up-regulated by Ad-hBMP-2 after the transfection at an increasing viral concentration in the dosedependent manner. Conclusion The results show that Ad-hBMP-2 can transfect the rabbit intervertebral disc cells in vitro with a high efficiency rate and the expression of hBMP-2 after theinfection is dose-dependent in the manner. AdhBMP-2 after transfection can up-regulate the expression of aggrecan and collagen Ⅱ mRNA at an increasing viral concentration.
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 study the effect of direct bone morphogenetic protein 2 (BMP-2) gene therapy mediated by adenovirus on repairing bone defect. Methods The radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly. The 4 groups were treated with different materials: group A, adenovirus carrying BMP-2 gene (AdBMP-2) plus bovine cancellous bone (BCB); group B, reconstructed BMP-2 plus BCB; group C, AdLacz plus BCB; and group D, only BCB scaffolds. The X-ray, histological examination, biomechanics analysis, and immunohistochemical staining were made 4, 8, and 12 weeks after the operation. Results Group A gained better effect in the volume of new bones, the anti-bending intensity of the healing bone, and the expression of BMP-2 than those of group B. The defect in group A was healed. No new bones were observed in group C and group D. Conclusion Direct BMP-2 gene therapy is easy to perform and has veryb osteoinduction ability. It is a good method to repair segmental bone defects.
Objective The expression of CD15 antigen and oncoprotein bcl-2 in thyroid cancer were examined in order to study the correlation between them. Methods The expression of CD15 and bcl-2 in 50 thyroid cancers, 20 adjacent noncancerous portion, 45 adenoma and 10 normal thyroid tissue were respectively investigated by microwave-LSAB immunohistochemical technique. Results The positive rate of CD15 and bcl-2 in thyroid cancer was 68.0% and 46.0% respectively, which was significantly higher than that in adenoma or adjacent noncancerous (Ｐ＜0.05). The percentage of CD15 and bcl2 positive expression were found to be significantly correlated with the tumor metastasis (Ｐ＜0.05), but not correlated with histological feature. Expression of CD15 was significantly correlated with bcl-2.Conclusion Expression of CD15 and bcl-2 can be regarded as a parameter to evaluate tumor metastasis and prognosis of thyroid cancer.