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.
【Abstract】ObjectiveTo construct a recombinant eukaryotic expression vector for human endostatin in order to study the inhibitory effect of liposome-mediated endostatin gene on the growth of human liver carcinoma in nude mice. MethodsHuman endostatin cDNA including IL-2 secreting peptide was cloned into eukaryotic expression plasmid pcDNA3.0 to construct recombinant plasmid pCD-sEndo. pCD-sEndo plasmid was transferred into hepatocarcinoma cell line SMMC-7721 mediated by liposome Dosper, the expression and secretion of endostatin gene was detected by RTPCR and Western blot analysis. The suspension of SMMC-7721 cells was injected subcutaneously at the back of 32 nude mice to establish the model of human liver carcinoma. The mice were divided into 4 groups randomly, and injected with Dosper+pCD-sEndo, Dosper+pcDNA3.0, Dosper and physiological brine separately. Tumor volume was measured by stages. The mice were executed after the drug had been given for 1 week, then the microvessel density (MVD) of the tumor tissue was detected with immunohistochemical method and apoptotic index of tumor cells was measured by TUNEL-stain. ResultsThe eukaryotic expression vector pCD-sEndo was successfully constructed and was confirmed by enzyme digestion and sequence analysis. Expression of endostatin gene was detected in transfected SMMS-7721 cells by RTPCR in vitro, and endostatin protein was also detected in the supernatant of transfected SMMS-7721 cells by Western blot. In vivo study, the growth of human liver carcinoma was inhibited in the group injected with endostatin gene: the average volume of tumor in this group was significantly smaller than that in other groups （P＜0.05）; the average MVD in this group was 6.2±2.5, significantly less than that in the group injected with physiological brine (32.8±6.4), Dosper （27.8±6.4）, or Dosper+pcDNA3.0 (25.5±5.5), P＜0.05. The average apoptotic index of tumor cells in treatment group, brine group, Dosper group and Desper+pcDNA3.0 group was 24.5±7.3, 7.6±2.5, 9.5±3.0 and 11.2±3.6 respectively, it was evidently higher in the treatment group than in the latter three groups. ConclusionHuman endostatin mediated by cation liposome could decrease the microvessel number of implanted human hepatocarcinoma in nude mice. It could also accelerate apoptosis of tumor cells and inhibit growth of tumor.
Objective To construct a replicationdefective recombinant adinovirus including the target gene human bone morphogenetic protein 4(fragment hBMP-4). Methods The hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into the eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into the competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/hBMP-4 was transformed into the packaging of thecell lines HEK293 to produce the replication-defective recombinant adenovirusescontaining the hBMP-4 gene. These replication-defective recombinant adinoviruses were transfected into HEK293 and HeLa cells. Then, total RNA and total protein were detected by RT-PCR and the Western-blot assay. Results The pAdE/hBMP-4 was confirmed by the restrictional endonuclease digestion. In HEK293 and HeLa cells, the specific transcription of the hBMP-4 gene was confirmed by RT-PCR, and the expression of the hBMP-4 protein was confirmed by theWestern-blot assay. Conclusion The replication-defective recombinant adinovirus expression vector containing the hBMP-4 gene can be constructed and expressed successfully, which has laid a foundation for the further research on the genetherapy of hBMP-4.
【Abstract】Objective To study the advances in clinical application of shape memory alloy (SMA) in general surgery. Methods The literature in the recent years on the advances of SMA was reviewed. Results At present clinical application of SMA have been developed. Different stents could dilate the benign and malignant stricture of blood vessels, biliary ducts, cola, recta and gastric outlet obstruction. The SMA catheter system was effective for peroral cholangiopancreatoscopy. Novel nitinol basket instrument might be useful for percutaneous cholecystolithotomy. Compression anastomoses was safe and sound in gastrointestinal surgery using a device made of SMA. Nitinol stent could be used in transjugular intrahepatic portosystemic stent shunt (TIPSS) with satisfactory results. Nitinol occlusion devices was superior to stainless steel coils. The Simon nitinol filter represented a new generation of venous interruption devices designed to prevent recurrent pulmonary embolism. Conclusion Clinical application of SMA will be increased by the development of endoscope, laparoscope and interventional technique.
Objective To investigate a change in the differentiation and biological function of the cultured rat fibroblast (FB) transfected by the myoblast determining gene (MyoD) and the connexin 43 (Cx43) gene and to explore the possible mechanism of the MyoD and Cx43 genes on treatment of ischemic heart disease (IHD). Methods The gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and pLenti6/V5DEST-Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. The RFL-6 FB cells were transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, followed by the Blasticidin (50 μg/ml) selection, according to the lentiviral expression system (ViraPower) protocol. The expression and the biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, and molecular and immunocytochemical methods. The mophological structure changes of the cells were observed under microscope before and after the transfection. Results The expression of MyoD and Cx43 was detected in the MyoD and Cx43 genes transfected FB with RT-PCR and Western blot. The immunocytochemical methods indicated the expressionsof the MyoD and Cx43 genes, while desmin and αactin were found in these cells. The myotubes were found from the cultures incubated a week in the differentiation medium, in which the transfected cells had a characteristic of the filamentsin their cytoplasm and showed a myoblast morphology. Conclusion MyoD cDNA can induce the cultured FB to differentiate into the myoblasts and Cx43 cDNA can enhance the gap junctional intercellular communication between the cell and the cell. Thus, a further experimental foundation for the therapy of IHD can be provided.
Non-directly affiliated hospitals are an important supplement to directly affiliated hospitals of medical colleges in China. Considering the problems of teaching consciousness, school running form and teachers construction in non-directly affiliated hospitals, this paper takes the medical personnel training of a non-directly affiliated hospital which is subordinate to Shenzhen Luohu Hospital Group as an example, in order to analyze the related exploration methods of non-directly affiliated hospitals under the development of hospital collectivization, and put forward a trinity training mode of general practitioners, which is “medical college-non-directly affiliated hospital-community health center”. This paper further discusses the challenges and possible solutions faced by non-directly affiliated hospitals in the new era.
Objective To introduce the studies on gene therapy for peripheral arterial disease(PAD) using plasmid DNA encoding human hepatocyte growth factor(HGF) gene. Methods Recent articles including preclinical and clinical studies were reviewed. Results Intramuscular injection of human HGF plasmid DNA into rat, rabbit, dog and diabetic hindlimb ischemic models, resulted in a significant increase in capillary density, blood flow and blood pressure. but no influence on tumor growth in mice. A clinical trial wasperformed in ischemic limbs of 6 critical limb ischemic patients, the result showed that no side effect caused by gene transfer was detected in all 6 patients.The pain scale and long diameter of ischemic ulcers were reduced. Conclusion Intramuscular injection of naked HGF plasmid DNA could be a safe and potential treatment for PAD.
Objective To investigate the effect of interleukin-10 （IL-10） gene transfer on expression of CD44, selectin-E, lymphocyte function associated antigen-1 （LFA-1）, vascular cell adhesion molecule-1 （VCAM-1） in mice heart transplantation rejection. Methods Model of mice cervical heterotopic heart transplantation was set up, 96 mice were divided into three groups with random number table, control group： heart transplantation between C57 mice; transplant group： heart from BALB/C mice transplant to C57 mice; IL-10 group： IL-10 was transfected on BALB/C mice isolated heart for 1 hour, then transplanted to C57 mice. The messenger ribonucleic acid （mRNA） level expression of CD44 ,selectin-E ,LFA-1 ,VCAM-1 and IL-10 were measured by reverse transcription-polymerase chain reaction （RT-PCR） at the 5th day after transplantation. Results The mRNA level expression of CD44, selectin-E ,LFA-1 ,VCAM-1 in transplant group were significantly increased than those in control group （P〈0.01）. The mRNA level expression of CD44, selectin-E, LFA-1 ,VCAM-1 in IL-10 group were significantly decreased than those in transplant group （P〈0.01）. Conclusion IL-10 gene transfer is able to decrease the expression of CD44, selectin-E,LFA-1 ,VCAM-1 and suppress the heart transplantation rejection in mice.