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.
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.
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: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.
Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
Objective To compare the differences of chromosome aberration and Rb 1 gene mutation among 3 cloned cells of SO-Rb50 cell line of retinoblastoma. Methods 1.Three cell cloned strains named MC2, MC3, MC4 were isolated from SO-Rb50. 2. Gbanding and karyotype analysis were performed on the llth passage cells of the 3 cell strains.3.All exons and the promoter region of the Rb gene were detected by PCR-SSCP analysis in tumor cell DNA extracted from the 3 cell strains. Results 1.Both numerical and structural chromosomal aberrations could be observed in these 3 cell strains.Several kinds of structural chromosomal aberrations were observed.The chromosome aberrations in the same passage of different cell strains were different.Aberration of chromosome 13 was rare and the aberration feature was different in the 3 cell strains.Five marker chromosomes were identified.M1,t(1;1)qter-p35∷q24-ter could befound in all cell strains.Two of them M4 and M5,have not been reported in SO-Rb50 cell line previously.2.SSCP analysis of exon 24 showed that MC411 and MC3138 had abnormal band. Conclusions The characteristics of heterogeneity of the original tumor cell line SO-Rb50are still kept during a long-term culture in vitro and the cloned strains had dynamic changes during this period.Aberration of chromosome 13 is not the only cause of RB;aberration of chromosome 1,a commom event in some neoplasias as well as in SO-Rb50, plays a meaningful role in the immortalization of this cell line. (Chin J Ocul Fundus Dis, 1999, 15: 146-148)
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 evaluate the host immune reaction against adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP-2) gene therapy in repairof tibial defects. Methods Twelve goats were made 2.1 cm segmental defects in he tibial diaphysis and divided into 2 groups. AdvhBMP2 transfected marrow mesenchymal stem cells(MSCs) and untransfected MSCs were implanted into the defect sites of transfected group(n=7) and untransfected group (n=5), respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. Results More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes (SI) increased 14 days after implantation, and there was significant difference between the transfected group (4.213±1.278) and the untransfected group(－0.310±0.147，Plt;0.05); SI decreased after 28 days, but there was no significant difference between the transfected group (2.544±0.957) and the untransfected group (3.104±0.644，Pgt;0.05). After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 (log0.1) were 2.359±0226, 2.297±0.200, 2.214±0.215 and 2.297±0.210 in transfected group, and -0.175±0.335, -0.419±0.171, 0±0.171 and 0.874±0.524 in untransfected group, being significant differences betweentwo groups(Plt;0.05). Conclusion Adenovirus mediated BMP-2gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.
Objective To investigate the current situation, problems of medicinal biotechnology in China, and to provide the relevant countermeasures for its development. Methods We surveyed the units which could carry out medicinal biotechnology projects in 30 provinces except Tibet, and compared the results with that in America.Results The questionnaire were returned from 25 provinces (83.4%), and there were 1 477 medicinal biotechnology projects carried out by 149 units in the past 10 years. These projects ranged from basic biotechnology to regenerative medicine and stem cell researches. The basic research projects constituted quite large percentage among all the projects. But the development levels in different areas were imbalanced, cross correlation with the development levels of economy. An echelon team of talents has been developed, most of them were trained in China. The invested capital differed considerably among units, in general the amounts were insufficient. Most invested capital came from the government. The number of patent application for projects based on independent-developed technology was small. This showed that project principals had a poor understanding of patents. More than half of units did not have a Bioethics Committee. From the search result for documents, the number of articles on stem research of China was close to that in America; and the number of articles on gene treatment and tissue engineering has already exceeded that of America. However, research on gene diagnosis of China was lagging far behind America. Conclusions An echelon team of talents has been developed, most of them are trained in China.We should give full play to the advantage of the distribution of qualified personal resources in developed economical areas so as to promote the applicability and popularity of medicinal biotechnology in less developed areas.Regarding to applicability and development, we should first develop applied technology to form the core competetiveness of basic research, technology development and application; we should also strengthen the training in ethics and regulation to establish a set of scientific assessment of medicinal biotechnology and management system.
Objective To systematically evaluate the effects of magnesium sulfate on postoperative pain and complications after general anesthesia. Methods A literature search was conducted in following databases as The Cochrane Library, EMbase, PubMed, EBSCO, Springer, Ovid, CNKI and CBM from the date of establishment to September 2011 to identify randomized controlled trials (RCTs) about intravenous infusion of magnesium sulfate during general anesthesia. All included RCTs were assessed and the data were extracted according to the standard of Cochrane systematic review. The homogenous studies were pooled using RevMan 5.1 software. Results A total of 11 RCTs involving 905 patients were included. The results of meta-analyses showed that compared with the control group, intravenous infusion of magnesium sulfate during general anesthesia significantly reduced the visual analog scale (VAS) scores at the time-points of 2, 4, 6, 8, 16, and 24 hours, respectively, after surgery, the postoperative 24 hours morphine requirements, and the incidents of postoperative nausea and vomiting (RR=0.61, 95%CI 0.40 to 0.91, P=0.02) and chilling (RR=0.29, 95%CI 0.14 to 0.59, P=0.000 7). Although the incidents of bradycardia (RR=1.93, 95%CI 1.05 to 3.53, P=0.03) increased, there were no adverse events or significant differences in the incidents of hypotension and serum concentration changes of magnesium. Conclusion Intravenous infusion of magnesium sulfate during general anesthesia may obviously decrease the pain intensity, and the incidents of nausea and vomiting and chilling after surgery, without increasing cardiovascular adverse events and risk of hypermagnesemia. The results still need to be confirmed by more high-quality and large-sample RCTs.