目的 通过检测人睾丸生殖细胞肿瘤中的Skp2蛋白质异常表达,探讨相关意义。 方法 应用S-P免疫组织化学法检测睾丸生殖细胞肿瘤，正常睾丸组织和慢性睾丸炎组织中Skp2的表达。 结果 睾丸生殖细胞肿瘤中Skp2阳性表达率为74.5%，正常睾丸组织中Skp2阳性表达率为20.0%，在慢性睾丸炎组织中Skp2阳性表达率为40.0%，在3种不同睾丸组织中表达差异有统计学意义（P＜0.05）；Skp2表达与不同组织学类型的睾丸生殖细胞肿瘤无相关性（P＞0.05）；随着临床分期的增高，睾丸生殖细胞肿瘤中的Skp2表达增多，差异无统计学意义（P＞0.05）。 结论 在人睾丸生殖细胞肿瘤中的Skp2高表达，提示细胞周期的异常调控在睾丸生殖细胞肿瘤的发生、分化中起着重要的作用。
Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
ObjectiveTo investigate the expression of Yes-associated protein (YAP) screened by bioinformatics in rats with myocardial-ischemia reperfusion injury and establish the base for further research. MethodsThe difference of gene spectrum of rats with myocardial-ischemia reperfusion injury was analyzed by bioinformatics technique. The related signaling pathways and key genes were screened by KOBAS2.0 and KEGG. Eighteen Sprague Dawley rats were randomly divided into three groups: normal group (n=6), sham operation group (n=6) and myocardial-ischemia reperfusion injury group (n=6). The expression of target gene was detected by immunochemistry, quantitive reverse transcription polymerase chain reaction and western blotting. ResultsA total of 345 differentially expressed genes were found by bioinformatics, among which 181 were up-regulated and 164 were down-regulated. The differential genes were mainly enriched in Wnt, HIPPO, MAPK, Jak-STAT and other signaling pathways. We focused on HIPPO pathway and found that the expression of YAP increased significantly in myocardial-ischemia reperfusion injury group, compared with the normal group and sham operation group (P＜0.05). ConclusionsThe expression of YAP of HIPPO signal pathway is increased in rats with myocardial-ischemia reperfusion injury.
Objective To investigate the effect of chondroitinase ABC (ChABC) on the expression of growth associated protein 43 (GAP-43) and gl ial fibrillary acidic protein (GFAP) after spinal cord injury (SCI) in rats. Methods A total of 150 adult female SD rats, weighing 250-300 g, were randomly divided into ChABC treatment group (group A), sal ine treatment group (group B), and sham operation group (group C) with 50 rats in each group. In groups A and B, the rats were made the SCI models and were treated by subarachnoid injection of ChABC and sal ine; in group C, the rats were not treated as a control. At 1, 3, 7, 14, and 21 days after operation, the Basso, Beattie, and Bresnahan (BBB) score system was used toevaluate the motion function, and immunofluorescent histochemical staining was used to observe the expressions of GAP-43 and GFAP. Results At different time points, the BBB scores of groups A and B were significantly lower than those of group C (P lt; 0.05); there was no significant difference in BBB score between groups A and B after 1, 3, and 7 days of operation (P gt; 0.05), but the BBB score of group A was significantly higher than that of group B after 14 and 21 days of operation (P lt; 0.01). At different time points, the GAP-43 and GFAP positive neurons of groups A and B were significantly higher than those of group C (P lt; 0.05). After 14 and 21 days of operation, the GAP-43 positive neurons of group A were more than those of group B (P lt; 0.01). After 7, 14, and 21 days of operation, the GFAP positive neurons of group A were significantly less than those of group B (P lt; 0.01). Conclusion ChABC can degrade gl ial scar, improve the microenvironment of the injured region and enhance the expression of GAP-43, which promotes axonal growth and extension.
Objective To observe the effects of neural stem cells(NSCs) transplantation on the glial cell line-derived neurotrophic factor (GDNF) and growth associated protein 43(GAP-43) after the spinal cord injury(SCI), and to investigate the mechanism of repairing the SCI by NSCs transplantation. Methods The neural stem cells from the hippocampus of rats’ embryo were cultured and identified by immunocytochemistry. The SCI model was made by the modified Allen device. Sixty adult Wistar rats were randomly divided into three groups: spinal cord injury was treated with transplantation of NSCs (group A, n=24), with DMEM solution(group B, n=24) and normal control group without being injured(group C, n=12). Seven days after the operation of SCI, the NSCs were transplanted into the injured site. Then GAP-43 and GDNF expressions were tested by RT-PCR and immunohistochemistry. Results Compared with group B, the GDNF mRNA expression of group A increased by 23.3％ on the 1st day, by 26.8% on the 3rd day and by 32.7% on the 7th day; the GAP-43 mRNA expression increased by 19.5% on the 1st day, 21.6% on the 3rd day and 23.1% on the 7th day. There were statistically significant differences(Plt;0.05). Conclusion The transplantation of NSCs can change the microenvironment injured site and promote the regeneration of axon by enhancing the expressions of GDNF mRNA and GAP-43 mRNA. It is one of the mechanisms of repairing the SCI by NSCs transplantation.
Objective To dynamically study the formation of multidrug resistance(MDR) of human hepatocellular carcinoma cell SMMC-7721 induced by Adriamycin (ADM) and the role of multidrug resistance-associated protein(MRP) in its mechanisms.Methods Hepatocellular carcinoma cell SMMC-7721 was cultured in RPMI-1640 medium containing ADM with progressively increased concentration or directly cultured in medium containing different concentrations of ADM. Resistant index of drug-resistant variants of SMMC-7721 cell was determined by drawing cell dosage-reaction curves.Levels of MRP mRNA expression were detected by reverse transcription-polymerase chain reaction(RTPCR). Intracellular rubidomycin(DNR) concentration was examined by flow cytometry(FCM).Results With progressive increasing of ADM concentration in medium resistant index and levels of MRP mRNA expression were correspondingly increased but intracellular DNR concentration was markly reduced. When parental cells were directly cultured in medium containing different concentrations of ADM, the higher the ADM concentration, the higher the level of MRP mRNA expression, but intracellular DNR concentration was kept at the similar high level and most cells died. Conclusion ADM may progressively induce SMMC-7721 cell resistant to multiple chemotherapeutic drugs with reduced intracellular DNR accumulation associated with the overexpression of MRP gene.
ObjectiveTo observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.MethodsPrimary cultured human müller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Müller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups.ResultsAll müller cells expressed glutamine synthetase (＞95%), which confirmed the cultured cells in vitro were the purification of generations of müller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human müller cells. qRT-PCR indicated that SP1 (t=28.30, P＜0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P＜0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.ConclusionProbucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in müller cells induced by high glucose.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.