Hua xi yi ke da xue xue bao = Journal of West China University of Medical Sciences = Huaxi yike daxue xuebao最新文献

Objective: To construct a recombinant BCG secretively expressing ESAT-6 of Mycobacterium tuberculosis.

Methods: alpha-antigen(alpha-Ag) signal sequence and esat-6 gene were amplified from the genome of Bacille Calmette-Guerin (BCG) and Mycobacterium tuberculosis by PCR respectively. esat-6 gene was cloned in E. coli-BCG shuttle-plasmid pMV261 to get pME. Then a new recombinant plasmid pSME was constructed by inserting BCG alpha-Ag signal sequence into pME.

Results: The cloned genes alpha-Ag signal sequence and esat-6 were correctly inserted into the vector pMV261, which was confirmed by restriction endonuclease digestion and PCR amplification of pSME.

Conclusion: pSME was expected to secretively express ESAT-6 of Mycobacterium tuberculosis in BCG. This study provides the possibility of further researches on the development of new anti-tuberculosis vaccine.

Objective: To explore the change in the expression of NT3 in the process of promoting the plasticity of spinal cord by acupuncture.

Methods: Five adult cats were subjected to unilateral spared root rhizotomy; their L1-L5, L7-S2 dorsal root ganglia (DRG) were sectioned, but L4 was spared. And two groups of acupoints [Zusani (St.36) and Xuanzhong (G. B.39); Futu (St.32) and Sanyingjiao (Sp.6)] located in hind limb were electro-stimulated for thirty minutes q.d. x 7. At seven days, after acupuncture, the L5 segment of spinal cord and spared dorsal root ganglion (L6) were taken and made into frozen section 20 microns in thickness. Immunohistochemistry (NT3 antibody 1:1500) and in situ hybridization (NT3 cRNA probe 1:100) techniques were used. The numbers of positive neuron for NT3 and it's mRNA in large, medium, small neuron of L6 DRG and the numbers of positive neurons and glia cells for NT3 in lamina II were counted respectively.

Results: The numbers of positive large, small neurons for NT3 and its mRNA in DRG and the number of positive neurons and glia cells for NT3 in lamina II on the acupuncture side increased apparently than those on the non-acupuncture side (P < 0.05). However, the positive signal of NT3 mRNA in lamina II was not seen in our study.

Conclusion: The results indicate that acupuncture promoting the plasticity of spinal cord involves both the increase in expression of NT3 in large and small neurons of spared DRG and the increase in number of NT3 positive neurons and glia cells in spinal lamina II. Moreover, NT3 may play a role in the process of promoting the plasticity of spinal cord by acupuncture.

Objective: The evolution processes of type IV collagen(CN), fibronectin(FN), and laminin(LN) in carcinogenesis were studied by the use of diethylnitrosamine induced rat hepatocarinogensis model so as to clarify the action of chemical carcinogens on the mechanisms of the occurrence and development of hepatocellular carcinoma.

Methods: Immunohistochemical technigue and image analysis were used to demonstrate the change of the above mentioned three extracellular matrices (ECMs) in histopathological foci.

Results: There were no ECMs expressed in the altered foci, whereas in some neoplastic nodules the ECMs were expressed intensively along the capillarized sinusoid. Cellular morphological analysis showed that in these nodules the average perimeter and the area of the cell increased significantly, but nucleus/cytoplasm ratio decreased significantly as compared with the controls. In the nodules with intensive expression of ECMs, cell proliferation was active, but in the hepatocellular carcinoma these three ECMs diminished or vanished.

Conclusion: The ECMs play an important role in the occurrence and development of hepatocellular carcinoma.

Objective: To understand the change and clinical significance of PEF in smokers, COPD and cor pulmonale patients.

Methods: The F-V curves were measured in 116 smokers, 90 COPD (43 with chronic bronchitis, 47 with emphysema) and 31 cor pulmonale patients in the ameliorated period. The indices selected were FVC, PEF, V75, V50 and V25.

Results: The PEF% (measured values/predicted values) in smokers and chronic bronchitis patients were higher than 84%(i.e. percentages being within the normal range); they were 44% and 32% in patients with emphysema and cor pulmonale respectively and decreased with the severity of disease. The [formula: see text] (%) in smokers and chronic bronchitis patients were 87.7%-89.7% (again, percentages being within the normal range); they were 70.7% and 41.9% in patients with emphysema and cor pulmonale respectively and decreased with the severity of disease. The phenomenon that decrease was more conspicuous in V75% than in PEF% could be explained with the wave-speed theory.

Conclusion: There is clinical significance in the degree of decrease in PEF% and [formula: see text] (%).

Objective: To demonstrate the change of pulmonary function in patients with end-stage renal disease(ESRD) before and after peritoneal dialysis (PD).

Methods: In this study were measured the forced vital capacity (FVC), maximum breathing capacity (MBC), the forced expiratory volume of the first second (FEV1), maximal expiratory flow (MEF), maximal mid-expiratory flow rate (MMEF), 25% of maximal expiratory flow (V25), and the diffusion of co in lung (DLco) for 50 patients with end-stage renal disease and 20 normal subjects. All the indexes were determined again in 30 ESRD patients two months after peritoneal dialysis.

Results: The indexes of pulmonary ventilation (FVC, MBC, FEV1, PEF, MMEF, V25) and the pulmonary diffusion DLco were lower in the ESRD patients than in the controls. FEV1, PEF, MMEF and V25 were improved markedly after peritoneal dialysis in ESRD patients (P < 0.05); FVC, MBC, and DLco were of no change (P > 0.05).

Conclusion: The function of pulmonary ventilation and diffusion are decreased in patients with ESRD accompanied with airways obstruction. Peritoneal dialysis can improve airways obstruction remarkably, but it has no effect on the function of pulmonary ventilation and diffusion.

Objectives: To improve the accuracy and precision of the determination of bilirubin, especially direct bilirubin (DB), and the standardization of that as well.

Methods: Purified conjugated bilirubin (Bc) and ditaurobilirubin(DTB) and their diazo products were subjected to absorption spectrum analysis. The diazo reaction characters of their calibration solutions were compared by the method of Doumas J-G(TB & DB).

Results: Bc, DTB and their azopigments were found to have the similar absorption spectra with the same lambda max. Their TB standard curves almost superposed together all over. Although the slopes of their DB standard curves were not markedly different ((YBc = 0.00366X + 0.00933, rBc2 = 0.9977, P < 0.01; YDTB = 0.00391X + 0.00023, rDTB2 = 0.9987, P < 0.01; Pb1-b2 > 0.05, n1 = n2 = 5), the DB value measured for Bc differed from that for DTB(n = 5, P < 0.05). In addition, the calibrators made from Bc based different matrices, such as HSA, BSA and human serum, were significantly different in DB/Bc, but no difference was seen among the concentrations. Furthermore, the DB values determined for DTB or Bc increased linearly with the corresponding concentrations, respectively, with no difference between the slopes (YBc = 0.8300XBc + 1.9463, rBc2 = 0.9977, P < 0.01; YDTB = 0.8853XDTB-0.0251, rDTB2 = 0.9986, P < 0.01; n1 = n2 = 5, Pb1-b2 > 0.05).

Conclusions: The results demonstrate that the diazo reaction characters of Bc are identified with those of DTB. However, under the condition of DB, Bc reacts differently from DTB. This study also indicates that as a calibrator of DB based human serum, Bc has the similar constant effect of HCl as serum samples do, so it is a more reliable calibrator to eliminate the matrix effects.

Objective: To investigate the structure and function of human nerve growth factor (beta-NGF) and the gene encoding beta-NGF.

Methods: A pair of specific primers (29 mer) for the sequence encoding human beta-NGF was designed and synthesized. A 380 bp fragment was amplified from human blood genomic DNA by polymerase chain reaction, and cloned into pGEM-T Easy vector. The identified insert fragment from the recombinant pGEM-T-NGF was directionally ligated with linearized pGEX-5T with the compatible termini. E. coli JM 109 was transformed with the expression recombinant p5TNGF and induced by IPTG.

Results: The cloned DNA fragment was identified as the full-length sequence encoding human beta-NGF by restriction analysis and DNA sequencing. SDS-PAGE and Western blot revealed the cloned NGF gene expressed as a fusion protein (40.5 x 10(3) u) in the cells transformed by p5TNGF. The soluble fusion protein was determined to be 503.2 mg/L, accounting for 6.8% of the total soluble protein (7.4 g/L) of bacterial cells. This fusion protein was found to have antigenic activities of NGF.

Conclusion: The clone containing the full-length sequence encoding human beta-NGF is obtained and successfully expressed in E. coli to be of use for studying the biological functions of human beta-NGF gene.

Objective: To get high quality and sufficient numbers of mature dendritic cells (DC) from healthy human peripheral blood.

Methods: Cultured plastic-adherent monocytes were isolated from healthy human peripheral blood by use of granulocyte-monocyte clony-stimulating factor and Interleukin-4 for 7 days without fed with fresh medium and cytokine.

Results: A large number of DCs with high purity were generated. These DCs expressed HLA-I, II molecules, Costimulating molecules and adherent molecules highly, showing the characteristics of mature DC. These DCs could stimulate proliferation of allogenic T lymphocytes and had active endocytosis ability which peaked at the third day in culture but decreased afterwards.

Conclusion: These results provide evidence that CD monocytes isolated from peripheral blood monocytes exhibit dendritic cells characteristics and may facilitate further studies of DC and its clinical application.

Objective: To gain an understanding of the expression of beta-NGF in myoblasts transfected by PSVCEP NGF-CAT and the transfecting efficiency of Lipofect AMINE.

Methods: PSVCEP NGF-CAT was delivered into cultured myoblasts by Lipofect AMINE. Expression of beta-NGF in the transfected myoblasts was detected by immunocytochemistry.

Results: Lipofect AMINE was evidently engulfed by myoblasts at the 6th hour; about 40% myoblasts could be detected with the expression of beta-NGF by immunocytochemistry at the 48th hour.

Conclusion: PSVCEP NGF-CAT can express beta-NGF in cultured myoblasts, and Lipofect AMINE is a convenient vector for gene transfection with simplicity and efficacy.

Objective: To establish a specific method for determination of IgG in IVIG.

Methods: The IVIG samples were disposed by acylation and analyzed by Rocket Immunoelectrophoresis. The amount of the IgG in IVIG was calculated by an IgG standard curve.

Results: The standard curve by this method has a very good linear relationship: r = 0.9967. The reproducibilities with 3 bathes of IVIG samples (n = 6) are 5.37% +/- 0.21%, 5.28% +/- 0.39% and 5.80% +/- 0.30%.

Conclusion: This method could be adapted to regular assay in blood products.