Objective To purify recombinant methioninase and investigate the synergetic inhibitory effect of methioninase and cisplatin on the proliferation of human lung adenocarcinoma cell line GLC. Methods Recombinant methioninase was purified with GST-column from supernatant after ultrasonic disruption of cultured Escherichia coli in the prokaryotic expression system pGEX-4T-1-Met/Dh5a. MTT assay was used to determine the inhibition rate of methioninase in combined with cisplatin on cell proliferation,and their synergistic effect was evaluated by using the q value judge method. Results The concentration of recombinant methionine cleaving enzyme was 0.22 mg/mL, the purity was 95%, and the activity was 0.568 IU/mg. After 72 h culturing, the inhibition rate of cisplatin methionine was 24.80%and 27.49%respectively,while the inhibition rate of the combined drugs was 66.80% ( =1.47>1.15) which showed a significant synergistic effect. Conclusion Both methioninase and cisplatin have the inhibition effect,and the combined drugs display a significant synergistic effect on the proliferation of GLC.

Objective: To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hTNF-?, investigate the antitumor effect of recombinant adenovirus. Methods: Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hTNF-?. Cell growth assay, colone formation test, flowcytometry assay and morphology were used to observe the effects on tumor cells. The hTNF-a gene, which was transduced into cancer cells mediated by recombinant adenovirus, was detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay. The intratumoral injection of rAd-LacZ and rAd-hTNF-? was carried out to evaluate their antitumor effects. Results: The liter of rAd reached 1010 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd. Except the surface structure and ultrastructure of tumor cells infected with rAd had a light change, cell growth abillity assay, colone formation test, flow cytometry assay showed no significant difference compared with that of the control cells. The TNF-? gene expression at 24 h increased greatly. Antitumor study indicated that on the tumor-bearing mice treated with rAd the tumor grew slowly. Tumor volume was significantly smaller and survive time was prolonged than that of controls. Conclusion: There was no significantly changes occurred on tumoral cells after infected with recombinant adenovirus expressing hTNF-?. The intratumoral injection of rAd-LacZ and rAd-hTNF-? could inhibit the growth of solid tumor.

Objective:To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hIL 2, and investigate the antitumor effect of the rAd Methods:Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hIL 2(rAd hIL 2) Cell growth assay, colony formation were used to observe the effects on tumor cells hIL 2 gene was transduced into cancer cells mediated by recombinant adenovirus, then detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay The intratumor injection of rAd LacZ, rAd hIL 2 was carried out to evaluate its antitumor effects Results:The titer of rAd reached 10 10 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd Growth inhibition assay, colony formation assay showed no significant difference compared with that of the control cells The 24 h IL 2 expression increased greatly Antitumor study indicated that the tumor grew slowly in the bearing tumor mice treated with rAd Tumor volume was significantly smaller and survive time was prolonged than that of controls Conclusion:There were no significantly changes of tumor cells infected with recombinant adenovirus expressing hIL 2 The intratumor injection of rAd LacZ?rAd hIL 2 could inhibit the growth of solid tumor

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; genetics ; DNA, Neoplasm ; genetics ; Melanoma, Experimental ; genetics ; pathology ; Mice ; Recombinant Proteins ; genetics ; Transduction, Genetic ; Tumor Cells, Cultured

Objective: To investigate in vitro the biological characteristics of AdCD80-infected human lung cancer cells on the basis of generation of replication-deficient hB7-1(CD80) recombinant adenovirus. Methods: Human CD80 gene was transduced into lung cancer cells mediated by recombinant adenovirus and then the expression of the gene was detected by PCR and agarose gel electrophoresis. The biological characteristics of the above cells were analysed with electron microscope, FACS and etc. Results: The titers of rAd reached to 10 10 PFU/ml and more than 90% Anip973 lung cancer cells could be infected by 30 MOI rAd. The growth curve and cloning efficiency of rAdCD80-infected Anip973 cells showed no significant difference compared with that of the control cells. The cell proliferation cycle of rAdCD80-infected 973 cells showed no change through FACS test. Having been infected by rAdCD80, the surface structure and ultrastructure of 973 cells had a little change. Conclusion: These results will lay foundation for tumor vaccines.

Methionine-dependent increase in tumor cells is a specific metabolic defect. This metabolic defect is also a target for selective treatment of cancer. Studies found that the methionine gamma-lyase (methioninase, L-methionine gamma-lyase) can specificly split the methionine of extracellular and intracellular, so it can strongly inhibit the growth of tumor cells and induce apoptosis of tumor cells. However, no effect on normal cells has been found, so the methionine gamma-lyase may play the anti-tumor role. We also explored in the present study another effect of methionine gamma-lyase as a single agent on DNA methylation levels and DNA synthesis, which may change as a result of deprivation of methionine, and thus may enhance anti-tumor effects. Animal studies and clinical trials showed that a variety of methionine dependent methionine gamma-lyase can eliminate the tumor cells. Therefore, methionine restriction is an effective anticancer strategy. Methionine gamma-lyase has shown great prospects as a new type of gene therapy. This article made a review of it.
Animals ; Carbon-Sulfur Lyases ; administration & dosage ; Genetic Therapy ; methods ; Humans ; Neoplasms ; drug therapy ; therapy ; Recombinant Proteins ; administration & dosage ; Transfection

OBJECTIVE： To optimize the expression induction condition of two recombinant methioninases， and to investigate their inhibitory effects on the proliferation of human lung adenocarcinoma cells GLC. METHODS： Recombinant methioninases expression plasmid PGEX-4T1-4A1-MGL and PGEX-4T1-3B8-MGL were transfected into competent Escherichia coli Dh5α， and induced by isopropyl-β-D-thiogalactoside. Using the expression level of target protein as index， the initial OD600 nm value before induction， culture temperature and induction time were optimized by single factor test. The recombinant methioninase 4A1-MGL and 3B8-MGL were purified by affinity chromatography. The concentration of recombinant methioninase was detected by Coomassie blue method. The purity of the product was detected by sodium lauryl benzene sulfonate-polyacrylamide gel electrophoresis； its activity was detected by spectrophotometry. The proliferation of cells was detected by MTT assay after treated with low-dose， medium-dose and high-dose of recombinant methioninases （4A1-MGL or 3B8-MGL was 0.1， 0.2， 0.4 U/mL） for 24， 48， 74 h. Inhibitory rate of cells were calculated. RESULTS： The optimal induction condition of two recombinant methioninases included that initial OD600 nm of 0.9， culture temperature of 37 ℃， induction time of 5 h. The results of validation test showed that protein expression level of 4A1-MGL was 1.52±0.04， that of 3B8-MGL was 1.28±0.03 （RSD＜3％，n＝3）. After purification， the concentration， purity and activity of 4A1-MGL were （0.70±0.02）mg/mL， （96.42±3.15）％ and （0.45±0.02）    U/mg； and those of 3B8-MGL were （0.56±0.02）mg/mL， （97.43±2.96）％ and （0.91±0.03）U/mg. After treated with low-dose and medium-dose of 4A1-MGL and 3B8-MGL for 48 and 72 h， treated with high-dose of 4A1-MGL and 3B8-MGL for 24， 48 and 72 h， inhibitory rate of GLC cell was increased significantly， and high-dose group for 72 h was significantly higher than low-dose and medium-dose groups at same time point （P＜0.05）. CONCLUSIONS： The induction conditions of recombinant methioninase expression are successfully optimized in this study. The obtained 4A1-MGL and 3B8-MGL could inhibit the proliferation of GLC cells in a dose-dependent manner.

Objective:To explore the risk factors of bladder recurrence in patients with upper urinary tract urothelial carcinoma (upper tract urothelial carcinoma, UTUC).Methods:We retrospectively analyzed the data of 815 patients underwent radical nephroureterectomy for upper tract urothelial carcinoma between June 2009 to June 2019.There were 519 males and 340 females, aged from 26-93 years old(average 66.5±9.6 years old). 396 patients were renal pelvic caicinoma.463 patients were ureteral caicinoma.675 patients were accompanied with hydronephrosis.664 patients were accompanied with preoperative gross hematuria. Preoperative diagnostic ureteroscopy was performed in 323 cases.283 patients had the history of smoking.48 patients were con-comitant with bladder carcinoma at the first diagnosis. Univariate analysis and logistic multivariate regression analysis were used to investigate the risk factors for bladder recurrence after UTUC radical surgery.Results:Among the 859 patients, 407 (47.4%) had low-stage tumor (T is/T a/T 1), 452 (52.6%) had high-stage tumor (T 2-T 4), 110 (12.8%) had low-stage tumor (G 1/G 2), and 749 (87.2%) had high-stage tumor (G 3). 126 (17.2%) of 859 patients had relapse during the follow-up period, the average follow-up time was 17 months, the median recurrence time was 12 months, 101(80.1%) of the relapse occurred within 2 years after operation. In univariate analysis, lower tumor stage ( P=0.047), higher tumor grade ( P=0.043), preoperative hematuria symptom ( P=0.023) and preoperative diagnostic ureteroscopy ( P=0.002) were closely related to bladder recurrence. Taking the above factors into the logistic multivariate regression analysis showed that tumor staging T is/T s/T 1 ( B=0.476, P=0.019), tumor grade G 3( B=0.848, P=0.024), preoperative hematuria symptom ( B=0.521, P=0.048), preoperative diagnostic ureteroscopy( B=0.521, P=0.002) were independent risk factors of postoperative recurrence of bladder. Conclusion:lower tumor stage, higher tumor grade, preoperative hematuria symptom and preoperative diagnostic ureteroscopy are the independent risk factors of postoperative bladder recurrence in patients with UTUC. Routine intravesical chemotherapy should be performed in patients with UTUC with the above risk factors, and routine diagnostic ureteroscopy is not recommended.

OBJECTIVE： To study the effects of processed Polygonum multiflorum containing serum on the proliferation and the expression of estrogen receptor （ER） of human breast cancer T-47D cells， and to investigate its phytoestrogen （PE）-like effect. METHODS： Sexually immature SD rats were randomly divided into estradiol valerate （Ev） group （positive control， 0.12 mg/kg）， processed P. multiflorum low-dose and high-dose groups （0.75， 3 g/kg， by crude drug）， low-dose and high-dose processed P. multiflorum+Ev groups （same dose as single drug group）， with 10 rats in each group. Blank group was given constant volume of water intragastrically， and administration groups were given relevant medicine intragastrically； once day and night， for consecutive 4 days. Two hours after last administration， blank serum and containing serum were prepared. T-47D cells were also randomly divided into blank group， Ev group， low-dose and high-dose processed P. multiflorum groups， low-dose and high-dose processed P. multiflorum+Ev groups， and then were cultured in medium which contained 20％ blank serum or drug containing serum. CCK-8 assay was used to detect proliferation rate （PR）. Western blotting assay and RT-PCR were used to detect the protein and mRNA expression of ER-α and ER-β. RESULTS： Compared with blank group， PR of administration groups [each administration group （24 h）， other administration groups （48， 72 h） except for high-dose processed P. multiflorum+Ev group] were increased significantly； high-dose processed P. multiflorum group （72 h） was significantly higher than Ev group， and low-dose processed P. multiflorum+Ev group （72 h） was significantly higher than the same-dose processed P. multiflorum group； high-dose processed P. multiflorum+Ev group （72 h） was significantly lower than the same-dose processed P. multiflorum group （P＜0.05 or P＜0.01）. Relative protein expression of ER-α in Ev group， high-dose processed P. multiflorum group and low-dose processed P. multiflorum+Ev group， relative mRNA expression of ER-α and protein expression of ER-β in administration groups， relative mRNA expression of ER-β in Ev group， low-dose processed P. multiflorum group and processed P. multiflorum+Ev groups were all increased significantly. Relative protein and mRNA expression of ER-α in Ev group were significantly higher than processed P. multiflorum groups and combination groups. Relative protein and mRNA expression of ER-β in Ev group were significantly lower than low-dose processed P. multiflorum+Ev group， but relative mRNA expression of ER-β was significantly higher than processed P. multiflorum groups and high-dose processed P. multiflorum+Ev group. Relative protein and mRNA expression of ER-α and ER-β in low-dose processed P. multiflorum+Ev group as well as relative mRNA expression of ER-β in high-dose processed P. multiflorum+Ev group were significantly higher than the same-dose processed P. multiflorum group. Relative protein and mRNA expression of ER-α in high-dose processed P. multiflorum+Ev group were significantly lower than the same-dose processed P. multiflorum group （P＜0.05 or P＜0.01）. CONCLUSIONS： The processed P. multiflorum containing serum can promote the proliferation of human breast cancer T-47D cells， and play the PE-like role through promoting protein and mRNA expression of ER-α and ER-β. However， the above effects are weaker than estrogen， and the combination of the two may antagonize the effect of estrogen.

【Objective】 To investigate the causes of abnormal decrease in maximum amplitude(MA) of thromboelastography(TEG) and its effect on prognosis by monitoring the changes of coagulation-related indexes in emergency trauma patients. 【Methods】 A total of 319 cases of trauma patients admitted to our hospital from September 2020 to September 2023 were retrospectively analyzed, and the coagulation-related indexes of 0 h and 24 h after admission were observed. According to the MA results, they were divided into normal MA group(>50 mm) and reduced MA group(≤50 mm) to compare the hemoglobin(Hb), platelets count(Plt), activated partial thromboplastin time(APTT), prothrombin time(PT), fibrinogen(Fib), thrombin time(TT), D-dimer(D-D), coagulation reaction time(R), clot formation kinetics(Angle), 30 min clot dissolution rate(Ly30), MA, thrombine-antithrombin complex(TAT) and plasminase-α2 plasminase inhibitor complex(PIC). The correlation between MA and fibrinolysis indexes in 319 trauma patients was analyzed. According to whether tranexamic acid(TXA) was used, the reduced MA group was divided into a TXA group and a non-drug group. The differences in the change of the above coagulation-related indexes, mortality rate and changes in blood product dosage were compared between the two groups. 【Results】 Compared with the normal MA group, Hb, Plt, Fib, diastolic blood pressure and GCS scores decreased, while heart rate, ISS score and mortality increased significantly in the reduced MA group(P<0.05). The R, PT and TT were prolonged significantly(P<0.05), and PIC and D-D increased significantly(P<0.05) in the reduced MA group. Correlation analysis found that MA had no correlation with Ly30, TAT and APTT, but was correlated with Angle(r=0.803), Plt(r=0.544), Fib(r=0.581), PIC(r=-0.443) and D-D(r=-0.343). Compared with the non-drug group, the change of Angle, MA and FIB in the TXA group increased significantly(P<0.05), while the change of PIC decreased(P<0.05). Cryoprecipitate and platelet transfusion in the TXA group reduced significantly(P<0.05), and red blood cell transfusion had a decreasing trend, but the difference was not significant(P>0.05). The mortality rate in the TXA group was reduced significantly(P<0.05). 【Conclusion】 Hyperfibrinolysis may be an important factor in the abnormal decrease of MA in emergency trauma patients. Treatment with TXA can improve its effect on MA, and reduce the transfusion of blood products and the patient mortality.