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）
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 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 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.
Objective To observe the inhibitory effects of local co-transfection of tissuetype plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNAASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNAASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcriptionPCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty.Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups(P＜0.01).The number of PCNA positive cells in the transplanted arteries in PCNAASODN, tPA and tPA+PCNAASODN groups were significantly lower than that of control group(P＜0.05，P＜0.01). The intimal areas and degrees of luminal stenosis of PCNAASODN, tPA and tPA+PCNAASODN groups were lower than those of control group(P＜0.05，P＜0.01), and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups(P＜0.05). Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA+PCNAASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.
ObjectiveTo explore the new gene therapy method for tumor, the recombinant Caspase3 gene (rcaspase3) eukaryotic expression plasmid was constructed by molecular biologic method. MethodsThe eukaryotic expression plasmid pcDNA3.1(+)/rCaspase3 was constructed by rearrangement of the large subunit and small subunit of Caspase3 and it was transfected into pancreatic carcinoma cells(PCⅡ). After being transfected, the expression of rCaspase3 mRNA in pancreatic carcinoma cells was detected by RTPCR and it’s apoptotic activity was detected by FCM. ResultsThe sequence of rCaspase3 showed that the recombinant molecules (rCaspase3) now had its’ small subunit preceding its’ large subunit. After pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/rCaspase3 by liposomes, a 894 bp strap was observed by RTPCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was found by FCM.ConclusionThe plasmid of pcDNA3.1(+)/rCaspase3 has been constructed successfully. rCaspase3 has apoptotic activity and can be used as target gene in gene therapy for pancreatic carcinoma.
Objective To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P gt; 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg · min) in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
Objective To evaluate the potential of specific mRNA marker keratin 19(K19) to detect micrometastasis by reverse transcriptase polymerase chain reaction (RT-PCR) .Methods One hundred and ninty four regional lymph nodes harvested from 6 cases of benign diseases, 4 cases of breast carcinoma, 5 cases of gastric carcinoma and 12 cases of colorectal carcinoma patients were examined by conventional pathology and amplifying tissue specific K19 mRNA by RT-PCR separately, then the two methods were compared with each other. Results None of the 34 lymph nodes which were pathological metastasis-negative from benign diseases expressed K19 mRNA by RT-PCR, all of the 28 regional lymph nodes which were pathological metastasis-positive from malignant cases showed trains of K19 mRNA by RT-PCR. Of the 132 lymph nodes which were pathological metastasis-negative from malignant cases, 11 lymph nodes were detected with micrometastasis by genetic diagnosis.Conclusion Genetic diagnosis of lymph node micrometastasis is more sensitive than conventional pathology and has diagnostic value and merits further study.