Objective:To construct an eukaryotic expression vector of enhanced green fluorescence protein(EGFP) gene driven by telomerase catalytic subunit(hTERT) gene promoter and observe the specific expression of EGFP in lung cancer cell lines.Methods:The 1100bp promoter fragment was obtained by enzyme digestion from a recombinant plasmid of pGL3-hTERTp containing the hTERT promoter.The hTERT promoter was then subcloned into the upstream of the report gene EGFP of pEGFP-1 without promoter.The expression vector pEGFP-hTERTp was successfully constructed.The vector pEGFP-N1 containing cytomegalovirus(CMV) promoter was used as a positive control.The vector pEGFP-1 without promoter was used as a negative control.The vectors were transfected into human lung cancer cell lines 95D,NCI-H446,A2,A549,LTEP-a-2,YTMLC and normal MRC-5 through lipofectamine respectively.EGFP expression was detected under the fluorescence microscope.Results:pEGFP-hTERTp was confirmed by enzyme digestion with correct result.That the EGFP expression was detected in all of eight lung cancer cells transfected with pEGFP-hTERTp,but not in MRC cells.By contrast,high intensity EGFP expression was observed in both lung tumor cells and normal cells,which were transfected with pEGFP-N1.Conclusion:The EGFP controlled by hTERT promoter can be expressed specifically in lung cancer cell lines.hTERT promoter may be used as an excellent regulation element in tumor-targeting gene therapy.

Objective:To investigate the protective effects of nuclear factor ?B inhibition on liver graft reperfusion injury during transplantation.Methods:Orthotopic liver transplantation using modified cuff technique was established in rats,animals were divided according to the grafts cold storaged in 4℃ Ringer's lactated solution with(PDTC group)or without 0.1 mol/L PDTC(control group)for 6 h.During the early stage of reperfusion,DNA binding activities of NF ?B in liver grafts were analyzed by EMSA(electrophoretic mobility shift assay),mRNA level of TNF ? and ICAM 1 by RT PCR,and activities of ALT and LDH were also detected.Results:NF ?B in liver grafts was activated at early stage of reperfusion during transplantation; PDTC treated liver displayed lower activation of NF ?B 1 h after reperfusion,whereas no difference was shown between 2 groups 6 h after reperfusion.Up regulation of TNF ? and ICAM 1 transcription,high level of ALT and LDH activities were observed in both groups during reperfusion,whereas the transcriptional up regulation and the activities of ALT and LDH in PDTC group were reduced compared with those of control group( P

Objective:The effect of Tonglin capsule was observed on type steblay interstitial nephritis of cavy.Methods: The cavy was immunized using the basement membrane of renal tubule from renal cortex of rabbit and Freund's complete adjuvant (FCA). The animal model of autoimmunity renal tubule interstitial nephrifis was made, The effect of Tonglin capsule on nephric pathological changes was observed. Results: ① The levels of creatinine (Cr) and blood uria nitrogen (BUN) in treatment groups were obviously lower than that in model group ( P

Objective To evaluate the capability of 18 F-FLT uptake and investigate the early radiation response of human colorectal cancer cells HCT116 exposed to 6 MV X-rays.Methods 3.7 kBq 18F-FLT was added to HCT116 cells with different cell numbers (1.0 × 105-1.5 × 106) and cultured with different times (36,60,84 h).The 18F-FLT uptake rate was measured with a γ-counter after exposed to different does of 6 MV X-rays (0,2,4,6,8 Gy) after 24,48,and 72 h of irradiation.Then the cell uptake inhibition rate,cell proliferation,and cell cycle phase were measured.Results The uptake rate of 18F-FLT in HCT116 was (18.97 ± 1.16)％.The 18F-FLT uptake inhibition rates at 24 h after different does of irradiation (2,4,6,8 Gy) were (32.10±0.02)％,(54.46 ±0.04)％,(62.74 ±0.04)％,and (65.81 ±4.81)％,respectively,which was positively correlated with radiation dose.Conclusions The 18F-FLT uptake rate of human colorectal cancer HCT116 cells could be used to evaluate the early radiation response.

Objective To investigate the relationship between the expression of ITGAV and the radiosensitivity of NSCLC cells. Methods The expression of ITGAV in NSCLC and its relationship to the prognosis of patients who received radiotherapy were analyzed using bioinformatics methods. Differences in radiosensitivity between radio-resistant cells and parent cells were verified by clone formation experiment, and the protein expression of ITGAV was detected by Western blot. The transfection efficiency of si-ITGAV was determined by Western blot and qRT-PCR analyses. The best ITGAV interference sequence was selected to transfect A549R and H1299R cells. Clone formation experiment and flow cytometry were used to detect clone formation, apoptosis and cell cycle of A549R and H1299R cells. Results The expression of ITGAV in NSCLC tissues was significantly higher than that in normal tissues (P<0.05), and NSCLC patients with high ITGAV expression had poor prognosis. The clonogenic ability of the si-ITGAV group was significantly lower than that of the negative control group at 4, 6, 8Gy irradiation (all P<0.05). After 6 Gy irradiation, the apoptosis of the si-ITGAV group was increased (P_H1299R<0.0001, P_A549R=0.0002), the proportion of G₂/M phase cells to A549-siITGAV and H1299R-siITGAV cells was higher than that in the negative control group (P_H1299R<0.0001, P_A549R=0.0007). Conclusion Interfering with ITGAV expression can increase the radiosensitivity of NSCLC.

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.
Animals ; Animals, Inbred Strains ; Base Sequence ; China ; Cloning, Molecular ; Galactosyltransferases ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; genetics ; metabolism ; Sequence Analysis, DNA ; Swine ; Swine, Miniature ; genetics ; Transfection

Objective:To explore the feasibility and safety of the clinical application of the diagnosis and treatment mode combining rapid frozen pathological examination of prostate biopsy tissue with radical prostatectomy.Methods:Suspected prostate cancer patients with PSA>10 ng/ml and PI-RADS score ≥4 in, Northern Jiangsu People's Hospital from April to September 2021 were collected. The included patients underwent mpMRI/TRUS image fusion-guided transperineal prostate targeted biopsy with 16G biopsy needle, 2-3 needles for biopsy, and rapid frozen pathological examination. Robot-assisted laparoscopic radical prostatectomy (RALP) was performed immediately for patients with prostate cancer with rapid freezing pathology. For undiagnosed prostate cancer, 18G biopsy needle for prostate targeted + systematic biopsy were used, 18-22 needles for systematic biopsy, and routine pathological examination. The baseline data, frozen pathological results, perioperative conditions, pathological results and follow-up data of all patients were collected.Results:Eleven patients were included in the study, the mean age of the patients was 69.9(66-73) years, the mean BMI was 22.8(19-26) kg/m 2, the mean PSA was 23.2(14.25-32.00), the mean prostate volume was 45(32-52) ml, mean PSAD 0.54(0.33-0.75). PI-RADS score was 4 in 3 cases and 5 in 8 cases; digital rectal examination was positive in 5 cases. All 11 cases underwent rapid freezing and the pathological results showed that: 9 cases were prostate adenocarcinoma, and RALP was performed immediately. The operation time was 111.5(96-126) min, the intraoperative blood loss was 78.9(55-105) ml, and the postoperative extubation time was 4.3(3.5-5.0) days, postoperative hospital stay 5.8(5.0-6.5) days. Postoperative pathology showed that Gleason score 3+ 4=7 in 1 case, 4+ 3=7 in 3 cases, 8 points in 4 cases, and 10 points in 1 case; 3 cases had positive resection margins, and 1 case had seminal vesicle invasion, the average number of dissected lymph nodes was 10.9 (8.5-14.0), and there was no tumor metastasis. Pathological T staging included 2 cases of T 2b stage, 5 cases of T 2c stage, 1 case of T 3a stage, and 1 case of T 3b stage. Two patients were undiagnosed by rapid freezing pathology, of which one was prostate adenocarcinoma with a Gleason score of 4+ 3=7, and then received RALP; the other one was prostate inflammation. 11 patients were followed up; the postoperative follow-up time was 3-7 months, with an average of 5.2 months. Among the 10 patients who underwent RALP, 8 patients recovered urinary continence 2 weeks after surgery, and all recovered within 2 months after surgery. Three patients with positive surgical margins were given regular androgen deprivation therapy in the second week after surgery. PSA did not drop below 0.1 ng/ml in patients with positive margins and seminal vesicle invasion 3 months after surgery. No complications of Clavien grade Ⅰ or higher occurred after operation and during follow-up. Conclusions:For patients with high suspicion of prostate cancer, rapid frozen pathological examination of prostate biopsy tissue is performed. RALP is performed immediately for patients with prostate cancer. The results show that this diagnosis and treatment model could be safe and feasible.

Systemic sclerosis (SSc) is an autoimmune rheumatic disease that is characterized by skin fibrosis with multi-organ involvement. In China, the standardized diagnosis and treatment for SSc is still lacking. Based on the diagnosis criteria and guidelines from China and abroad, Chinese Rheumatology Association developed the current standardization of diagnosis and treatment for SSc. The purposes of this guideline are to standardize clinical management for SSc in China, to interpret the key evaluation tools for SSc, and to recommend therapeutic principle and strategies.

Objective:To investigate the effect of local anesthesia in patients with a PI-RADS score of 5 and ECOG score ≥2 for prostate puncture.Methods:Retrospective analysis of case data of 33 patients admitted to the Subei People's Hospital for prostate puncture from April 2020 to April 2022. Age (82.5±3.6) years. There were 18 cases with hypertensive disease, 8 cases with diabetes mellitus, and 6 cases with both diabetes mellitus and hypertensive disease. Body mass index (25.2±3.5) kg/m 2. prostate-specific antigen (PSA)(131.5±69.7) ng/ml. prostate volume (38.5±21.4) ml. all patients had a PI-RADS score of 5 on multiparametric magnetic resonance (mpMRI) and an Eastern Cooperative Oncology Group (ECOG) score ≥2. All 33 cases in this group underwent trans-perineal targeted prostate puncture using local anesthesia at the tip of the prostate. The visual analog score (VAS) and visual numeric score (VNS) were applied by the same surgeon to assess the patient's pain level and satisfaction at the time of puncture (VAS-1 and VNS-1) and 30 min after puncture (VAS-2 and VNS-2), and to record the duration of the procedure and the occurrence of postoperative complications. Results:In this group of 33 cases, the VAS-1 score was (1.9±0.3) and the VAS-2 score was (0.1±0.2); the VNS-1 score was (2.9±0.2) and the VNS-2 score was (3.9±0.1). Postoperative pathological results indicated that one of the 33 patients had a negative puncture result (pathology report indicating interstitial inflammation), while the rest of the patients had a positive puncture pathology report (puncture pathology report indicating prostate cancer), with a positive rate of 97%. One case of postoperative carnal haematuria occurred, which gradually improved after the patient was advised to drink water and take alpha-blockers. No perineal hematoma occurred, and all patients did not suffer complications such as urinary tract infection, urinary retention, azoospermia, vagal reaction, and infectious shock.Conclusion:In patients with a PI-RADS score of 5 and ECOG score ≥2, the use of single-hole local anesthesia for performing trans-perineal targeted puncture biopsy has the advantages of good paroxysmal pain and high safety.