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 To probe the possibility of direct transfer of exogenous gene into peripheral nerve and its following expression in vivo. METHODS The PCMV beta plasmid containing cytomegalovirus (CMV) promoter and Escherichia Coli (E. Coli), beta-Galactosidease (beta-Gal) structural gene (lacZ gene) was constructed and injected into the rabbit sciatic nerve. The control group was injected PBS solution. The injected nerves were sampled and tested by beta-Gal enzyme activity assay of the 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and beta-Gal histochemical stain. RESULTS In the control group, no beta-Gal enzyme activity was detected in the different stages after operation, and beta-Gal histochemical stains showed positive. In the experimental group, enzyme activity could be detected from 2 days to 30 days after operation, and the histochemical stains showed negative. CONCLUSION The exogenous gene can be transferred into peripheral nerve and expressed with bioactivity, thus the gene therapy to accelerate the recovery of nerve is practical.
ObjectiveTo introduce the relationship between the apoptosis hepatocyte and its genic mediation and the ischemia of portal vein. MethodsThe combination of related literatures and our research findings were made.ResultsPortal vein ischemia may induced hepatocyte apoptosis, p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. Expression of p53 gene is enhanced in hepatic tissue when hepatocyte apoptosis is not obvious, but after 24-72 h of portal vein ischemia, when hepatocyte apoptosis is obvious, enhanced expression of p53 gene or reduced expression of bcl2 gene occur. There exists close relationship between portal vein ischemia and hepatocyte apoptosis. Conclusion Apoptosis hepatocyte is involved in organic atrophy after ischemia of portal vein, and p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. At present, the mechanism of apoptosis of hepatocyte induced by ischemia of portal vein is not clear, which needs further study.
In order to observe the role of genetically modified Schwann cell (SC) with pSVP0Mcat in the regeneration of injured spinal cord, the cells were implanted into the spinal cord. Ninety SD rats were used to establish a model of hemi-transection of spinal cord at the level of T8, and were divided into three groups, randomly, that is, pSVP0Mcat modified SC implantation (Group A), SC implantation (Group B) and without cell implantation as control (Group C). After three months the presence of axonal regeneration of the injured spinal cord was examined by means of horseradish peroxidase (HRP) retrograde labelling technique and stereography. The results indicated that HRP labelled cells in Group A and B could be found in the superior region of injured spinal cord and the brain stem such as the red nuclei and oculomotor nuclei. The density of ventral hom neurons of the spinal cord and the number of myelinated axons in 100 microns of the white matter was A gt; B gt; C group. In brief, the pSVP0Mcat modified SC intraspinal implantation could promote regeneration of the injured spinal cord.
Objective To determine the association between the geneti c polymorp hisms of vascular endothelial growth factor (VEGF) gene and the prognosis for retinopathy of prematurity (ROP) in Chinese. Methods Twenty infants with threshold ROP who had undergone retinal photocoagulation were in the treated group and 20 infants with self-regressed ROP without any treatment were in the control grou p . In the two groups, all the infants had oxygen-breathing history and the sex a n d gestational age were all suitable to be compared, except birth weight. Polymer ase chains reaction-restriction fragment length polymorphism was used to determine the frequencies of VEGF genes in the two groups. Results The frequencies of +405C allele were higher in the treated group than those in the control group (P＜0.05). The frequencies of the VEGF-460T/C and +936C/T ploymorphisms were similar in both groups (P＞0.05). Conclusions The +4 05C/G ge netic polymorphisms of VEGF may correlate to the prognosis of ROP. The carriers of +405CC allele are more susceptible to ROP.
ObjectiveTo summarize the role of survivin gene in tumor. MethodsThe research status on biological characteristics the role of survivin gene in tumor for gene therapy and clinical application was retrospectively analyzed after related domestic and foreign literatures were reviewed. ResultsSurvivin gene was by far found to be the best powerful apoptosis inhibition gene, which played antiapoptosis role mainly through inhibiting directly the activity of caspase-3 and caspase-7 in the downstream of cascade reaction. Survivin gene promoted tumor cell proliferation and differentiation through speeding up tumor cells transition of G1→S phase and eluding the recognition of tumor cells to the apoptosis in G2/M phase. Survivin gene played important role in the intermediate links of vasiformation through angiogenic factor (VEGF, bFGF, Ang-1, and COX-2). ConclusionSurvivin gene may inhibit the apoptosis, promote the proliferation and differentiation of tumor cells and tumor angiogenesis, suggested that survivin gene has potential to act as a novel tumor marker and become an indicator of malignant tumor.
【Abstract】Objective To study the antitumor activity of HSVtk/CD combinative gene toward human cholangiocarcinoma in vivo. Methods Nude mouse models with transplanted subcutaneous cholangiocarcinoma were constructed and divided into 4 groups randomly, each group had 8 mice. Adenovirus solution free from suicide gene was injected in subcutaneous tumors of each mouse of control group. Adenovirus solution containing cytosine deaminase (CD), thymidine kinase (tk) and HSVtk/CD fusional gene were injected into single suicide gene either HSVtk or CD was transinfected into the tumor cells by injecting viras into subcutaneous tumor of mice of CD gene,tk gene and fused CD and tk gene group respectively. 24 hours after the injection, 5fluorocytosine (5FC) and ganciclovir (GCV) were injected introabdominally in each mouse. Growth of the tumors were monitored.Results Tumor growth of the genetransfection groups was suppressed in different degrees. Compared with the control group, the suppressing rates of the genetransfection groups were 41.2%, 55.7% and 70.0% respectively (P＜0.05). Histological examination showed good tumor growth in the control group, and tumor necrosis in the other 3 groups, particularly obvious in the group transfected with pAd(HSVtk/CD).Conclusion Combinative gene system has a b antitumor effect on cholangiocarcinoma in vivo. But it’s not powerful enough to eliminate tumor thoroughly because of insufficient “Bystander effect”.
The choice of genetic models was main difficulty in the meta-analysis of gene-disease association studies. In this study, we made a further discussion about the genetic model-free approach that proposed by Minelli et al. The program that coded by JAGS and R was carried out to perform the Bayesian procedure. In a real example, several kinds of prior distribution were used, including non-informative prior distribution and external clinical prior information. Especially, compared to Minelli’s study, we introduced clinical prior information. The results indicated that the pooled results were rather robust no matters the prior distribution were non-informative or informative, especially when the number of included studies were large.