【Abstract】ObjectiveTo construct a mrp1 expression vector and investigate its biological characteristics in HepG2 cells in vitro. MethodsThe 6.5 kb multidrug resistanceassociated protein （MRP) cDNA obtained from plasmid pGEM-mrp1 was cloned into the pCI-neo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated, and the mrp1 mRNA and MRP in these HepG2 cells were detected by means of RT-PCR and FCM respectively. ResultsThe mrp1 expression vector was established successfully, and the stable MDR hepatocarcinoma cell line (HepG2/mrp1) was developed as well. The content of the specific fragment of mrp1 mRNA was (56.8±6.37)% and MRP was 7.89 in the HepG2/mrp1 cells, the corresponding value in HepG2 cells was (9.67±3.26)% and 0.79 respectively. The difference was statistically significant (P＜0.05). ConclusionIt is practicable to establish MDR hepatocarcinoma cell line by transferring mrp1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
ObjectiveTo investigate the significance of expression and correlation of pyruvate kinase M2 (PKM2) and yes association protein (YAP) in hepatocellular carcinoma (HCC) tissue, and then explore the relationship between the 2 kinds of protein. MethodsA total of 120 patients' HCC tissues and adjacent tissues were collected retrospectively, who treated in our hospital from Apr. 2010 to Oct. 2013, the expressions of PKM2 and YAP protein in these HCC tissues and adjacent tissues were detected by SP immunohistochemical method, and then analyzed the relationship between the expressions of PKM2 and YAP ptotein with the clinicopathological features of HCC. Of the 120 patients, the expressions of YAP and PKM2 protein and its mRNA in 50 cases of HCC tissues and adjacent tissues were also examined by Western blot and real-time PCR methods respectively. Results① The immunohistochemical results showed that, the positive rate of PKM2 protein and YAP protein in HCC tissues were 67.50% (81/120) and 71.67% (86/120) respectively, which were both higher than those of adjacent tissues[PKM2 protein:20.83% (25/120); YAP protein:29.17% (35/120)], P < 0.050. ② The expression of PKM2 protein was significantly positively correlated with the expression of YAP protein in HCC tissues (r=0.519, P < 0.001). ③ In HCC tissues, the expression of PKM2 protein was significantly correlated with the diameter of tumor, TNM staging, differentiation of HCC, and lever of alpha fetal protein (P < 0.050), and the expression of YAP protein was significantly correlated with the differentiation of HCC and lever of alpha fetal protein (P < 0.050). ④ Western blot results showed that, the expression levels of PKM2 protein and YAP protein in HCC tissues were 1.25± 0.11 and 1.08±0.10 respectively, which were significantly higher than those of adjacent tissues (PKM2 protein:0.38±0.01, YAP protein:0.41±0.02), P < 0.050. ⑤ Real-time PCR assays results showed that, basing the expressions of PKM2 mRNA and YAP mRNA in adjacent tissues (both as 1), expressions of PKM2 mRNA and YAP mRNA in HCC tissues were 11.38±0.35 and 19.96±0.48 respectively, which were both higher than those of adjacent tissues (P < 0.050). ConclusionPKM2 and YAP protein were related to the initiation of HCC, and they were also closely correlated with the differentiation and prognosis of HCC.
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.
Objective To study the expressions of SKP2 and p27 in gastric carcinoma and pericancerous tissues and to detect the relationship between their expressions and clinicopathological features. Methods Forty-nine cases of gastric carcinoma spicemen and 20 cases of tissue adjacent to the carcinoma were cut and made into paraffin-embedded slices. The expressions of SKP2 and p27 were then detected by SP immunohistochemical method. Results The positive expression rate and score of SKP2 were both significantly higher in the gastric carcinoma tissues than those in pericancerous tissues (P＜0.01), whereas those terms of p27 were higher in pericancerous tissues (P＜0.05, P＜0.01). It was observed that the pericancerous tissues with positive SKP2 expression or with negative p27 expression showed atypical hyperplasis ranging from moderate to severe degrees. The positive rate and score of SKP2 were significantly lower in the cases of infiltrating depth T1+T2， without-metastasis of lymph node, with-metastasis of the first site lymph node, and without-metastasis of distant organs than those in infiltrating depth T3+T4, with-metastasis of lymph node, with-metastasis of the second or third site lymph node and with-metastasis of distant organs in gastric carcinoma tissues (P＜0.05， P＜0.01), whereas the results were contrary for p27 (P＜0.01). There was a negative correlation between the score of SKP2 and that of p27 in gastric carcinoma tissues (r=-0.65, P＜0.01). Conclusion The expressions of SKP2 and p27 may act as important biological markers to reflect carcinogenesis, progression, biological beheviors and prognosis of gastric carcinoma.
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.
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.
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 effect of different mechanical tensions on the expressions of RhoA/Rho associated protein kinases (ROCK) in rat tendon stem cells (TSCs). MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged, 2-3 months; weighing, 200-250 g) by enzymatic digestion method and cultured for 2-3 passages, then seeded on micro groovdishes. The 4% (4% stretch group) and 8% (8% stretch group) mechanical stretching was performed for 4 hours every day at 1 Hz. After 1, 2, and 3 days, the protein and mRNA expressions of RhoA and ROCK were measured by Western blot and real-time quantitative PCR. The cell proliferation was measured by cell counting kit 8. The cells were not stretched as control group. ResultsThe TSCs at passage 2 showed a cobble-stone shape and aggregation growth; TSCs seeded on micro groovdishes showed random growth, and the cells grew along the stretching direction after mechanical stretching. The mRNA expressions of RhoA and ROCK in control group, 4%, and 8% stretch groups showed an increasing tendency at 1, 2, and 3 days, showing significant difference between groups (P<0.05). The protein expressions of RhoA and ROCK in 4% and 8% stretch groups were similar to those in control group at 1 day (P>0.05), but the expressions in 4% and 8% stretch groups showed an increasing tendency at 2 and 3 days, which were significantly higher than those in control group (P<0.05). The cell proliferation of 8% stretch group was significantly lower than that of 4% stretch group and control group at each time point (P<0.05), but no significant difference was found between 4% stretch group and control group (P>0.05). ConclusionThe expressions of RhoA and ROCK of rat TSCs are positively correlated with stretch intensity. So RhoA/ROCK may be an important molecule in TSCs after mechanical stretching.
ObjectiveTo investigate biofilm formation on the surface of silica gel by breast surgery clinical specimens of Staphylococcus epidermidis and to analyze the relationship between biofilm formation and icaA, icaD, and accumulation-associated protein (aap) gene. MethodsBetween December 2011 and January 2013, 44 strains of Staphylococcus epidermidis were isolated from the clinical specimens of the female patients who had no symptom of infection. The icaA, icaD, and aap genes were detected by PCR and 4 genotypic groups were divided:icaA+icaD+/aap+ group (group A), icaA+icaD+/aap- group (group B), icaA-icaD-/aap+ group (group C), and icaA-icaD-/aap- group (group D). Biofilms mass was semi-quantified by semi-quantitative adherence assay after 8, 12, 24, 30, and 36 hours of incubation. The thickness of biofilms was measured by confocal laser scanning microscope (CLSM) at 12 and 24 hours after incubation. The ultrastructure of biofilms was observed by scanning electron microscope (SEM) at 24 hours after incubation. ResultsPCR test showed that 13 strains were icaA+icaD+/aap+(group A), 12 strains were icaA+icaD+/aap-(group B), 16 strains were icaA-icaD-/aap+(group C), and 3 strains were icaA-icaD-/aap-(group D). In 29 strains which had bacterial biofilm formation (65.9%), there were 13 strains in group A, 7 strains in group B, 9 strains in group C, and 0 in group D. The result of semi-quantitative adherence assay showed no significant difference in the absorbance (A) values among 4 groups at 8 hours (P>0.05). The A values of groups A, B, and C were significantly higher than that of group D at 12-36 hours, and group A was significantly higher than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The results of CLSM showed that the thickness of biofilm in groups A, B, and C was significantly larger than that in group D at 12 and 24 hours after incubation (P<0.05), and the thickness of biofilm in group A was significantly larger than that in groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The result of SEM showed that the mature biofilm could be observed on the surface of silica gel in groups A, B, and C, and the ultrastructure of biofilms in group A were the most abundant and extensive among 3 groups. The ultrastructure of biofilm in group B was similar to that in group C. No obvious biofilms formed in group D. ConclusionicaA, icaD, and aap genes all play key roles in the process for biofilm formation of Staphylococcus epidermidis. Futhermore, aap gene enhance the ability of biofilm-forming when aap and ica genes coexist, so the biofilm-forming ability of icaA+icaD+/aap+ is strongest.