2004年 · 第117卷第04期

Background This study was to review the distribution and shifting trend of fungal of culture specimens isolated from eyes of patients at the Beijing Institute of Ophthalmology, Tongren Hospital, Beijing, China. Methods The fungal culture-positive rate, the distribution and change of isolates of 2609 specimens collected in a 12-year period (1989 -2000) were retrospectively analyzed. Results In 775 positive cultures, 707 specimens (91.2%) were from the cornea, 22 (2.8%) from the conjunctiva, 15 (1. 9%) from the anterior chamber, 9 (1.2%) from the vitreous body, 3 (0. 4%)from the lacrimal sac, and 19 (2. 5%) from other parts of the eye. The average culture-positive rate was 29.7%. The ratio of the positive cultures in the first half year (from January to June) to those in the second half (from July to December) was 1: 2. 1. The main genus cultured was Fusarium sp(58. 7%), followed by Aspergirum sp (16. 8%). The percentage of Fusarium sp was increased from 53. 6%(1989 - 1994) to 60. 2%(1995 -2000), whereas the percentage of Aspergirum sp was decreased from 22. 3%(1989-1994) to 15.1%(1995 -2000). Conclusions Fusaruim sp is one of the most predominant pathogens of ocular fungal infection in northern China and its incidence tends to increase, but that of Aspergirum sp to decrease. It is very important to recognize the distribution and shifting trend of pathogenic fungi in the diagnosis, prevention and treatment of fungal keratitis. Chin Med J 2004; 117(4): 598-600

Background Corticosteroids remain the most effective therapy available for asthma. They have widespread effects on asthmatic airway inflammation. However, little is known about the effects of corticosteroids on the production of bone marrow inflammatory cells in asthma. This study observed the effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD34+ hematopoietic cells, so as to explore the possible effectiveness of a bone marrow-targeted anti-inflammatory strategy. Methods Balb/c mice were sensitized and challenged with ovalbumin (OVA) to establish an asthmatic model. For two consecutive weeks, asthmatic mice were challenged with OVA while being given either prednisone, montelukast, prednisone plus montelukast, or sterile saline solution. The mice were killed 24 hours after the last challenge with OVA, and bronchoalveolar lavage fluid (BALF),peripheral blood, and bone marrow were collected. Eosinophils in peripheral blood and BALF, and nucleated cells in BALF, peripheral blood, and bone marrow were counted. The percentages of CD34+cells, CD4+ T lymphocytes and CD8+ T lymphocytes among nucleated cells in peripheral blood and bone marrow were counted by flow cytometry. Immunocytochemistry and in situ hybridization were employed to detect expression of CD34 and interleukin (IL)-5Rα mRNA (CD34+ IL-5Rα mRNA+ cells)among bone marrow hematopoietic cells. Results Compared with the sterile saline solution group, the number of eosinophils in BALF and peripheral blood, CD34+ cells in peripheral blood and bone marrow, and CD34+ IL-5Rα mRNA+ cells in bone marrow of mice from the prednisone and prednisone plus montelukast groups were significantly lower (P <0. 01). The number of eosinophils in BALF from the montelukast group was also significantly lower (P<0. 05). Conclusions The results suggest that, in this asthmatic mouse model, prednisone probably inhibits proliferation, differentiation, and migration of CD34+ cells in bone marrow, blocks eosinophilopoiesis in bone marrow, and interferes with eosinophil migration into peripheral blood and subsequent recruitment in the airway. In addition, montelukast may suppress eosinophil infiltration into the lungs of asthmatic mice. However, a significant inhibitory effect of montelukast on the proliferation and migration of CD34+ cells and a cooperating effect with prednisone on bone marrow of asthmatic mice were not observed. Chin Med J 2004; 117(4): 592-597

Background In response to the inflammatory reaction, circulating leukocytes aggregate and adhere to the endothelial cells and eventually pervade into tissues, resulting in cell damage. This study was to detect the inflammatory reactions in mouse focal cerebral ischemia and their distinct characteristics in the ischemic basal ganglia and surrounding cortex. Methods Mice were subjected to permanent occlusion of the left middle cerebral artery (MCAO) by introducing a suture for 2 to 120 hours. The expression of intercellular adhesion molecule 1 (ICAM-1)and Mac-1 was determined immunohistochemically. The myeloperoxidase (MPO) activity of the ischemic regions was measured. Results Four hours after MCAO, the number of ICAM-1 positive vessels in the ischemic basal ganglia increased (9. 2 ±2. 8 per mm2), peaked at 48 hours (29. 6 ±4. 8 per mm2), and decreased after 72 hours. In the ischemic cortex, the number increased rapidly 4 hours after MCAO (19. 4 ±6.1per mm2), peaked at 48 hours (44. 4 ± 16. 8 per mm2), and declined after 72 hours. Mac-1 positive cells were seen in the ischemic basal ganglia (3. 4 ±1. 2 per mm2) 12 hours after MCAO, peaked after 48 hours (20. 2 ±6. 3 per mm2), and decreased after 72 hours. In the ischemic cortex, however, the number increased 4 hours after MCAO (4. 3 ± 1. 7 per mm2), peaked after 48 hours(20. 9 ±8. 4 per mm2), and remained high at 120 hours. The MPO activity increased in the ischemic basal ganglia 12 hours after MCAO (0. 111 ±0. 023 U/g), peaked after 24 hours (0.194 ±0. 059 U/g), and decreased after 72 hours. In the ischemic cortex, the MPO activity increased 12 hours after MCAO (0.110 ±0. 032 U/g), peaked after 24 hours (0.210 ±0.067 U/g), and remained elevated at 120 hours. Conclusions The increased expression of ICAM-1 in the ischemic brain of mouse in the early phase of MCAO followed by the over-expression of Mac-1 and the increased MPO activity suggests that focal ischemia leads to early onset of inflammation. The inflammatory response is more persistent and intensive in the ischemic cortex than in the ischemic basal ganglia. Chin Med J 2004; 117(4): 586-591

Background In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering. Methods DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope(SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated. Results DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation. Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs. Conclusion dynamic seeding of hBMSCs is a promising technique in bone tissue engineering. Chin Med J 2004; 117(4): 576-580

Background The iron catalyzed lipid peroxidation plays an important role in the autodestruction of the injured spinal cord. This study was to detect the antioxidation of melatonin against spinal cord injury(SCI) in rats. Methods Sity Sprague-Dawley rats were randomly divided into four groups: group A (n=15) for laminectomyanly, group B (n=15) for laminectomy with SCI, group C (n=15) for SCI and intraperitoneal injection of a bolus of 100 mg/kg melatonin, and group D (n=15) for SCI and intraperitoneal injection of saline containing 5%ethanol. The SCI of animal model was made using modified Allen's method on T12, Six rats of each group were sacrificed 4 hours after injury, and the levels of free iron and malondialdehyde (MDA) of the involved spinal cord segments were measured by the bleomycin assay and thiobarbituric acid (TBA) separately. Functional recovery of the spinal cord was assessed by Modified Tarlov's scale and the inclined plane method at 1, 3, 7, 14, 21 days after SCI. The histologic changes of the damaged spinal cord were also examined at 7 days after SCI. Results After SCI, the levels of free iron and MDA were increased significantly and the modified Tarlov's score and inclined plane angle decreased significantly in groups B and D. In group C, the Tarlov's score and inclined plane angle were increased significantly at 7, 14 and 21 days, with histological improvement. Conclusion: Melatonin can reduce the level of lipid peroxidation and prevent damage to the spinal cord of rat. Chin Med J 2004; 117(4): 571-575

Background Peptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells. Methods Heteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay. Results PNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99. 4% after 7 days; the rate of cloning inhibition was 98. 2% after 8 days;whereas for oligonucleotide groups, at the same concentration, the percentages were 50.1%and 67.5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P <0. 05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P <0. 05). At the same time, the impact on cell morphology was observed. Conclusions The results showed that PNAs are potent antisense reagents. The telomerase-associated therapies are very promising for the treatment of malignant tumours. Chin Med J 2004; 117(4): 566-570

Background Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165(VEGF165) gene in adeno-associated virus (AAV) gene delivery system. Methods Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hind III, and the downstream of angiopoietin 1contained restriction enzyme site BamH I . The upstream of VEGF165 contained restriction enzyme siteBgl II, and the downstream of VEGF165 contained restriction enzyme site BamH I. Using the multiple cloning sites (MCS) in plasmid pZero + + such as BamH I, Bgl II, Hind III, Not I, Xho I,Xba I, Sal I, BspH I, Ksp I and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS. Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion,which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietinl and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes. Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease. Chin Med J 2004; 117 (4): 562-565

Background It has been shown that the presence of leptin is associated with deabefes, glucose wefabolism and insulin metablism. In this research, we evaluated the presence of the leptin C-2549→A polymorphism in the Chinese population in Chongqing and verified its association with plasma leptin levels and anthropometric, metabolic, and clinical parameters. Methods Two hundred and sixty-nine patients with diabetes, 135 non-diabetic first-degree relatives of the patients, and 85 healthy controls were screened for the presence of C-2540 →A polymorphism using a PCR-RFLP assay. Body mass index, fasting leptin, fasting insulin, fasting glucose and homeostatic model assessment for insulin resistance (HOMA)-IR were also determined. Results In the type 2 diabetes group, AA genotype frequency (6. 32%) and A allele frequency(34. 94%) was higher than in normal controls (1.18%and 25.29%, respectively). Diabetic patients with the AA genotype had lower fasting leptin and insulin levels than those with other genotypes. Carriers with the AC genotype had decreased fasting leptin and insulin levels and longer duration of disease as compared with those with CC genotype. The HOMA-IR of patients with AA or AC genotypes was lower than those with the CC genotype. In non-diabetic relatives group, individuals with the AA genotype had a lower fasting leptin level than those with the AC genotype. The fasting insulin and HOMA-IR level of carriers of the AA or AC genotype were lower than those of the CC genotype, Conclusion The C-2549→A polymorphism in the leptin gene is associated with fasting leptin in patients with type 2 diabetes. The distribution of the genotypes in diabetic subjects from diabetic pedigrees differs from those in normal controls. The A allele frequency in diabetic patients is higher than that in normal controls. The haplotypes defined by genotypes are different in the familial subjects. Chin Med J 2004; 117 (4): 558-561

Background Von Hippel-Lindau (VHL) disease is a heraditary cancer syndrome caused by germline mutations of the VHL tumor on the suppressor gene. This study was to show the clinical characteristics of a large Chinese kindred with von Hippel-Lindau disease and to evaluate the role of the genetic test of VHL disease in the diagnosis of VHL disease and clinical screening of members of the VHL disease family. Methods DNA extracted from peripheral blood was amplified by PCR to three exons of the VHL gene in 27 members of a large kindred with VHL disease. PCR products were directly sequenced. The involvements of multi-organs in the kindred with VHL disease were confirmed by history taking and radiography. Results Of 47 members in the four generations of the kindred, 18 members were diagnosed as having VHL desease. Clinical manifestations of 18 patients included: central nervous system (CNS)hemangioblastoma (5), renal cell carcinoma and CNS hemangioblastoma (3), renal cell carcinoma and retinal angioma (3), renal cell carcinoma and multiple pancreatic cysts (1), renal cell carcinoma and retinal angioma and multiple pancreatic cysts (2), renal cell carcinoma and CNS hemangioblastomas and multiple pancreatic cysts (1), and multiple pancreatic cysts and multiple renal cysts (1), multiple pancreatic cysts (2). The common lesions of the 18 patients were renal cell carcinoma (55. 6%), CNS hemangioblastoma (50.0%), retinal angioma (27. 8%), and multiple pancreatic cysts (38. 9%). Among the 27 members who volunteered for genetic analysis, 15members including 9 affected family patients and 2 asymptomatic patients and 4 carriers, who are still alive, presented a codon 78 from Asn to Ser change at nucleotide 446 (A→G) in exon 1. Four members were carriers with the same VHL gene mutation. Two asymptomatic patients were initially diagnosed by genetic testing and subsequently confirmed radiologically and surgically. Members without gene mutation had no clinical evidence of VHL disease. Conclusions The large Chinese kindred with VHL disease was classified as type I. The main characteristics in the kindred were higher incidence of renal cell carcinoma and lower incidence of retinal angioma. Genetic test plays an important role in early detecting asymptomatic patients and the carriers in clinical screening of members of the families with VHL disease. It is also important to prevent the transmission of VHL disease to their offsprings in the kindred. Chin Med J 2004; 117(4): 552-557

Background Intrarenal activation of the renin angiotensin system (RAS) plays an important role in mediating renal fibrosis. Both angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II(Ang II) receptor antagonists have been shown to exert a protective role against diabetic and non-diabetic nephropathy. However, the exact mechanism of how blocking local RAS prevents renal fibrosis is unclear. The present study was to investigate the influence of a new Ang II receptor antagonist, irbesartan (Irb), on Ang II -induced hypertrophy in human proximal tubular cell line (HK-2). Methods The cell line, HK-2, was grown in Dulbeccos's Modified Eagle's Medium containing 10%heat-inactivated fetal calf serum. After rested in serum-free medium for 24 hours, the effects of Irb on Ang II (10-7 mol/L)-induced [3H]-leucine incorporation, total protein content (measured by the Coomassie brilliant blue G250 method), and change in cell size (determined by scanning electron microscopy) were observed. The influence of Irb on the cell cycle was analyzed by fluorescence activated cell sorter (FACS) flow cytometry. Results Ang II induced cell hypertrophy in a time and dose dependent manner. Stimulation of cells with Ang II for 48 hours resulted in a increase in [3H]-leucine incorporation [0 hour: (5584 ±1016)cpm/105 cells vs 48 hours: (10741 ± 802) cpm/105 cells, P <0.05], which was significantly attenuated by treatment with Irb. Ang II significantly increased the total protein content in HK-2 cells[control; (0.169 ±0.011) mg/105 cells vs Ang II group: (0.202±0.010) mg/105 cells, P<0.05], which was also markedly inhibited by cotreatment with Irb (P <0. 01). Scanning electron microscopy showed that Ang II induced an increase in average physical cell size, which was significantly inhibited by Irb [control: (11.92 ±1.62) μm; Ang II group: (20. 63 ±3.83) μm; Ang II + Irb group: (13. 59 ±3. 15) μm; P <0.01 vs control, respectively]. Furthermore, flow cytometry revealed that Ang II arrested cells in the G0-G1 phase, which was significantly reversed by treatment with Irb [G0-G1 cells in Ang II group: (76. 09 ±1. 82)%, in Ang II + Irb group; (67. 00 ±2. 52)%, P<0. 05]. Conclusion Irb can inhibit Ang II -induced hypertrophy in HK-2 cells. Chin Med J 2004; 117(4): 547-551