【Abstract】ObjectiveTo study the expressions of matrix metalloproteinase 2 (MMP2) and carbohydrate antigen 50 (CA50) in colorectal carcinoma, cancer-adjacent mucosa （2 cm from the nether edge of tumor）, cancerdistant mucosa （5 cm from the nether edge of tumor） and normal colorectal mucosa, and to elucidate their effects on the development of colorectal carcinoma. MethodsThe expressions of MMP2 and CA50 were detected immunohistochemically in 40 cases of colorectal carcinoma, cancer-adjacent mucosa, cancer-distant mucosa and 10 cases of normal colorectal mucosa. Results①The expression intensity and positive rates of MMP-2 and CA50 increased significantly in turn by normal mucosa, cancer-distant mucosa, cancer-adjacent mucosa and colorectal carcinoma. ②The expression of MMP2 was correlated with CA50 in colorectal carcinoma. ③The expression of CA50 in colorectal carcinoma was closely associated with tumor differentiation, and the expression of MMP2 in colorectal carcinoma was closely associated with differentiation and Dukes stages as well. ConclusionOver expression of MMP2 facilitates the malignant progress of colorectal carcinoma; CA50 is a reliable marker of malignance in colorectal carcinoma; CA50 and MMP2 may have synergetic effects on the development of colorectal carcinoma.
Objective To evaluate the activity of the pancreatic tissue phospholipase A2 (PLA2) in acute pancreatitis (AP) and the therapeutic effects of verapamil in rats. MethodsThe model of rat AP induced by a closed duodenal loop technique was established to observe the changes of PLA2 activity in AP group and treated group. The pancreatic histology was examined by light and electron microscopy. Results At 16 and 24 hours after induction of AP in rats, significant inhibition of the pancreatic tissue PLA2 activity was shown in the treated group as compared with AP group, with 32.34±3.87u, 35.26±4.52u and 44.83±5.31u, 47.77±5.86u respectively. The treated animals also showed a decrease in the severity of pancreatic hemorrhage, necrosis and damage to the cellular ultrastructures. Conclusion There exists high activity of PLA2 in rats AP. Calcium channel blocker, verapamil might take therapeutic effects on AP by inhibiting activity of PLA2.
The activities and distributions of succinate dehydrogenase(SDH),malic dehydrogenase(MDH),lactic dehydrogenase(LDH),acid phosphatase(ACP) and alkaline phosphatase(AKP) in retinal vessels were studied and observed with enzymatic histochemical techniques. The retinal vessels showed a b LDH activity, moderate SDH and MDH activity. The dehydrogenase activity described above was evenly and equally distributed in the microvasculature between arterioles and venules, and was the best in arteries. AKP showed predominant activity in the endothelial cells of capillaries and arterioles which were stained bly. No activity for ACP observed in the retinal vessels. The observations above indicate that the retinal vessels are metabolically active and have a great capacity for glycolysis. （Chin J Ocul Fundus Dis,1992,8:6-9）
ObjectiveTo investigate the effect and mechanism of gastric bypass surgery (GBP) on fasting bloo-glucose (FBG) in type 2 diabetic rats. MethodsThe models of type 2 diabetic rats were induced by stretozotocin and 20 diabetic rats were randomly divided into two groups: diabetes-operation group (DO group, n=10) and diabetes-control group (DC group, n=10). Another twenty normal rats were randomly divided into two groups: normaloperation group (NO group, n=10) and normal-control group (NC group, n=10). The rats underwent GBP in DO group and NO group and sham operation in DC group and NC group. The FBG levels, serum dipeptidyl peptidase Ⅳ (DPPⅣ), and glucagon-like peptide-1 (GLP-1) concentrations of rats in each group were detected before operation and at 72 h, on 1 week, 4 weeks, and 8 weeks after operation. ResultsThe FBG levels of rats before operation were not significantly different between DO group and DC group or between NO group and NCgroup (Pgt;0.05). After operation, the FBG levels of rats in DO group gradually declined, reached the bottom on 4 weeks after operation and rose slightly on 8 weeks; The FBG levels of rats in DO group were lower after operation than before operation (Plt;0.05); After operation the FBG levels of rats in DO group were higher than that in NO group and NC group at the same time point (Plt;0.05); In DC group, the difference of FBG levels of rats at different time point was not statistically significant (Pgt;0.05); The inter-group and intra-group difference of FPG levels of rats for NO group and NC group was not statistically significant (Pgt;0.05). The concentrations of serum DPP-Ⅳ of rats before operation were not significantly different in each group (Pgt;0.05). After operation, the concentrations of serum DPP-Ⅳ of rats in DO group and NO group gradually decreased and markedly lower than that before operation, respectively (Plt;0.05). The concentrations of serum DPP-Ⅳ of rats after operation in DO group and NO group were significantly lower than that at the same time point in DC group and NC group, respectively (Plt;0.05); The intragroup difference of serum DPP-Ⅳ concentrations of rats for DC group and NC group was not statistically significant (Pgt;0.05). The concentrations of serum GLP-1 of rats before operation were not significantly different between DO group and DC group or between NO group and NC group (Pgt;0.05). After operation, the concentrations of serum GLP-1 of rats in DO group and NO group gradually increased, reached the top on 4 weeks after operation and declined slightly on 8 weeks; The concentrations of serum GLP-1 of rats in DO group and NO group were higher after operation than before operation (Plt;0.05);After operation, the concentrations of serum GLP-1 of rats in NO group were higher than that in NC group (Plt;0.05), but the concentrations of serum GLP-1 of rats at different time point in NO group were not different (Pgt;0.05). The intragroup difference of serum GLP-1 concentrations of rats for DC group and NC group was not statistically significant (Pgt;0.05). ConclusionsThere is obvious hypoglycemic effect of GBP on FBG levels of type 2 diabetic rats other than normal rats, in which high secretion of GLP-1 and low secretion of DPP-Ⅳ may be play an important role.
Objective To identify and isolate the variant gene associated with gastric adenocarcinoma and clone the fragment of variant gene.Methods By arbitrarily primer polymerase chain reaction (AP-PCR), DNA samples from 5 matched gastric adenocarcinoma and non-tumor gastric tissues were analysed. Results The produced AP-PCR profiles were different in each matched gastric adenocarcinoma and non-tumor gastric tissue. One differentiated amplified DNA fragments PW2.2 from a matched gastric adenocarcinoma were cloned. The result of Southern blot hybridization with PW2.2 as a probe showing that this fragment was also found in some other gastric adenocarcinoma samples. Conclusion AP-PCR fingerprinting assay can be used to identify and clone the variant genes associated with gastric adenocarcinoma.
ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
Objective To evaluate which is better method zymogen or low temperature frozen in removing vascular endothelial cell so as to lay a foundation for creating a kind of brace which is not to be rejected and the same as own blood vessel. Methods Fresh and not damaged umbilical blood vessel was collected from natural labour women, human umbilical blood vessel was remove carefully from normal foetus, then was put into disinfectant at 37℃ for 24 hours. They were divided into 3 groups:normal group(NG),zymogen group(ZG) and low temperature frozen group(LG). ZG: 0.1% collagenⅡ enzyme was addedin umbilical blood vessel and closed the both sides and the vascular endothelialcell was removed in 37℃ water. LG:Umbilical blood vessel was put into liquidnitrogen for 24 hours after frozened step by step, and then it was put into 37℃ water for 30-60 s and the vascular endothelial cells were washed away by normal saline. NG:Umbilical blood vessel was kept into 4℃ Kerb’s liquid. The bacteria were culturedin each group. The samples were stained by HE,elastic fiber and collagen fiberwere observed by light and scanning electron microscope. The difference of compliance was compared. Human leukocyte antigen ABC(HLA-ABC) and HLA-DR were observed by immunohistochemical method and the expression of antigen of umbilical blood vessel was analysed. Results In LG, umbilical vascular endothelial cells were removed completely; artery showed vertical smooth muscle and vein showed elastic membrane. InZG, umbilical vascular endothelial cells were removed completely after 20 minutes;artery showed vertical smooth muscle cells and vein showed lower endothelial layer. The vascular compliance in LG was higher than that in NG, and the latter was also higher than that in ZG,but showing no significant differences (Pgt;0.05). The compliance of umbilical vein was 2-3 times as much asthat of umbilical artery.The expression of HLA-ABC and HLA-DR in LG andZG were lower than that in NG, showing significant differences (Plt;0.01). Conclusion Low temperature frozen methodand zymogen method(0.1% collagen Ⅱ enzyme for 20 min) can remove vascular endothelial cells of human umbilical blood vessel completely.Low temperature frozenmethod was better than zymogen method.
Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
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.
OBJECTIVE: To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. METHODS: Fibroblasts from children’s foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. RESULTS: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen. CONCLUSION: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.