ObjectiveTo investigate the diagnostic value of internal medicine thoracoscope combined with pleural GeneXpert MTB/RIF for tuberculous pleurisy.MethodsEighty patients with tuberculous pleurisy admitted to hospital with pleural effusion were treated as tuberculous pleurisy group, and 20 patients with clinical diagnosis of malignant pleural effusion were used as control group. After admission to the hospital, the pre-operative examination of internal medicine thoracoscope were analyzed. All patients were extracted pleural effusion with thoracic puncture in order to send pleural tuberculosis smear and culture. Patients who had no contraindications were arranged internal medicine thoracoscope to get pleural effusion which will be sent to GeneXpert MTB/RIF and pathological tissue biopsy.ResultsIn the tuberculous pleurisy group, nine patients were positive in pleural tuberculous smear, and the positive rate was 11.3%; 4 patients were positive in pleural tuberculous culture, and the positive rate was 5.0%; 75 patients were diagnosed with pathological biopsy, and the positive rate was 93.8%; 69 patients were positive with pleural GeneXpert MTB/RIF, and the positive rate was 86.3%. The positive rate of internal medicine thoracoscopic pleural biopsy combined with pleural GeneXpert MTB/RIF could reached 96.3%. The pleural GeneXpert MTB/RIF lifampin resistance gene was positive in 5 patients, 4 of them were positive for tuberculosis culture, and the drug sensitivity results showed rifampicin resistance. In the control group, patients had negative result in pleural effusion tuberculosis smear, tuberculosis culture and the pleural GeneXpert MTB/RIF.ConclusionsThe diagnosis of tuberculous pleurisy by the combination of internal medicine thoracoscope and pleural GeneXpert MTB/RIF has high specificity and sensitivity. The diagnosis of tuberculous pleurisy by the combination of internal medicine thoracoscope and pleural GeneXpert MTB/RIF has high specificity and sensitivity, which has the value of rapid and accurate diagnosis and early guidance of anti-tuberculosis chemotherapy based on the early judgment of whether rifampin resistance exists.
The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E.coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28%±1.45%(P<0.05)and 75.50%±2.63%(P<0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SY5Y cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.
Objective To investigate the clinical application value of GeneXpert Mycobacterium tuberculosis (MTB)/ rifampin (RIF) in urine samples for tuberculosis diagnosis. Methods The patients with clinically highly suspected tuberculosis admitted to West China Hospital of Sichuan University between January 1, 2018 and June 1, 2023 were selected retrospectively. The diagnostic efficacy of urine GeneXpert MTB/RIF detection, such as sensitivity, specificity, positive predictive value, and negative predictive value, were retrospectively analyzed to evaluate its clinical value in the diagnosis of tuberculosis. Correlation analysis was further conducted to explore the correlation between positive levels of GeneXpert MTB/RIF in urine samples and laboratory test indicators. Results A total of 400 patients were included. Among them, 163 cases were in the clinical tuberculosis group and 237 cases were in the clinical non tuberculosis group. In the clinical tuberculosis group, 112 cases were urogenital tuberculosis patients and 51 cases were non-urogenital tuberculosis patients. The sensitivity, specificity, positive predictive value, and negative predictive value of urine GeneXpert MTB/RIF in the diagnosis of tuberculosis were 55.2%, 97.5%, 93.8% and 76.0%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of urine GeneXpert MTB/RIF in the diagnosis of urogenital tuberculosis were 65.2%, 92.0%, 76.0% and 87.2%, respectively, and the diagnostic sensitivity was further improved. Correlation analysis showed that the positive degree of urine GeneXpert MTB/RIF was correlated with the levels of hemoglobin, serum total protein, blood serum albumin, and other indicators. Conclusions Urine GeneXpert MTB/RIF detection offers high sensitivity and specificity in the diagnosis of tuberculosis, especially in urogenital tuberculosis, which is helpful for the early and rapid diagnosis of tuberculosis patients. The positive degree reported by the GeneXpert MTB/RIF in urine may indicate disease severity.
Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
Objective To review the research progress of the treatment of osteosarcoma, and to thoroughly understand its current state of research and prospect so as to lay a sol id foundation for the cl inical treatment. Methods The cl inical and experimental research l iteratures about treatment of osteosarcoma were extensively reviewed and analyzed. Results The present treatment of osteosarcoma is still need to comprehensive therapy which combine chemotherapy and surgical treatment. There are some progresses in gene therapy and molecular targeting therapy which can improve survival rate. Furthermore, well-designed studies and cl inical trials are needed to evaluate the potential therapeutic impact before they are used in cl inical. Conclusion Advancement in chemotherapeutic regimens has improved survival and l imb-sparing surgery in the treatment of osteosarcoma, but the progress of gene therapy and molecular targeting therapy gives new hope for osteosarcoma patients.
Despite its low incidence, retinoblastoma and its rela ted gene (Rb gene) have attracted some of the most brilliant minds in medicine and biology fi elds over the past years. Great advances have been achieved in the tumoregenesis mechanism and clinical management of retinoblastoma recently. However as always , more questions arise from those results. In order to improve retinoblastoma re search in China, we need to strengthen the communication and cooperation with di ffe rent countries, different institutes and disciplines, and utilize the great reso urces of retinoblastoma patients in China.
Objective To explore the feasibility of recombinant adeno-associated virus (rAAV) as a vector for the gene therapy of liver cancer. Methods The rAAV/enhance green fluorescein protein (EGFP) recombinant was prepared by the routine method of two plasmids cotransfection.Results The experiment showed that one 10cm plate could produce 107-108 infection unit recombinant by the method of two plasmids cotransfection, and the transduction of HepG2 cell was increased with the increase of infection dosage of rAAV. About 100 multiplicity of infection (MOI) AAV vector could make all the tumor cell light. Conclusion Liver cancer cell can be efficiently transduced by rAAV, and AAV vector may be a valuable vector for the gene therapy of liver cancer.
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 investigate the effect of B7-1 and IL-12 gene expression on the immunogenicity of hepatocellular carcinoma (HCC) HepG2 cells. Methods Plasmids encoding B7-1 and IL-12 molecules were retrovirally introduced into human HCC cells,empty vector as control. PBLs were cocultured with HepG2/B7-1,HepG2/IL-12 and HepG2/neo cells. Three days later,PBLs were submitted to specific cytotoxicity test and nonspecific cytotoxicity test against K562 cells by MTT assay.Results HLA-Ⅰ molecules on PBLs were detected by FACS.HLA-Ⅰ molecules expressing on PBL cocultured with HepG2/B7-1,HepG2/IL-12 cells were enhanced by 16.95% and 14.71% than those of HepG2/neo group, respectively(Ｐ＜0.05). Specific cytotoxicity against HepG2/B7-1 cells was 12.5% higher than that of against HepG2/neo cell,while no increase in that of against HepG2/IL-12 cells. Cytotoxicities against K562 cells in HepG2/B7-1,HepG2/IL-12 groups were 19.38% and 14.78% higher than those of HepG2/neo group, but no significant difference between the first two groups.Conclusion B7-1 and IL-12 gene transfer could remarkably promote immunogenicity of hepatocellular carcinoma cells and induce b specific and nonspecific immunity against hepatocellular carcinoma in vitro.