2005年 · 第118卷第01期

Background Antigen-loaded eosinophils (EOSs) instilled intratracheally into mice were capable of inducing Th2-type cytokine production in the draining lymph nodes. The aim of the present study was to evaluate whether EOSs within the tracheobronchial lumen can stimulate Th2 cell expansion in the lung tissues. Methods Airway EOSs were recovered from ovalbumin-sensitized and -challenged BALB/c mice, these EOSs were then cocultured with CD4+ cells isolated from sensitized mice in the absence or presence of anti-CD80 or/and-CD86 monoclonal antibodies. Airway EOSs were instilled into the trachea of sensitized mice. At the day 3 thereafter, the lung tissues were removed and prepared into cell suspensions for culture. Cell-free culture supernatants were collected for detection of cytokines. Results Airway EOSs functioned as CD80- and CD86-dependent antigen-presenting cells to stimulate lung CD4+ lynphocytes to produce interleukin-4, interleukin-5 and interleukin- 13, but not interferon-γ in in vitro assay. When instilled intratracheally in sensitized recipient mice, airway EOSs primed lung Th2 cells in vivo for interleukin-4, interleukin-5 and interleukin-13, but not interfeon-γ, production during the in vitro culture that was also CD80- and CD86-dependent. Conclusion EOSs within the lumina of airways could process inhaled antigen and function in vitro and in vivo as antigen-presenting cells to promote expansion of Th2 cells in the lungs. Chin Med J 2005; 118(1): 6- 11

Background It is still unclear whether viral genetic variability influences response to interferon (IFN)-α treatment. Recent reports suggest that IFN-α effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-1ranscriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-α treatment. Methods The HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and conparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into Hep G2 cells, which were then exposed to recombinant human cytokines. Results The T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patient s. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-α/γand tumor necrosis factor (TNF)-α except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-α on HPRE. Conclusions There are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-α. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-α other than that of IFN-γor TNF-α on HPRE. Chin Med J 2005; 118(1):56-61

Background Recently, arsenic trioxide (AS2O3) was considered as a novel anti-tumor agent. However, it showed severe toxicity effect on normal tissue at the same time. To improve its therapeutic efficacy and decrease its toxicity, we prepared arsenic trioxide-loaded albuminutes immuno- nanosheres [AS2O3- (HAS-NS)-BDI-1] targeted with nonoclonal antibody (McAb) BDI-1 and tested its specific killing effect against bladder cancer cell. Methods AS2O3-HAS-NS was prepared by chemical cross-linking method. Monoclonal antibody BDI-1 was purified with ammonium sulphate saltingout and chromatography. Albuminutes microspheres were conjugated with McAb by SPDP cros-linking method. Concentration of As in AS2O3-(HAS-NS)-BDI-l and AS2O3-HAS-NS was measured by atomic fluometry method. AS2O3-(HAS-NS)-BDI-l and its activity were detected by SDS-PAGE reduction electrophoresis, indirect immunofluorescence test, light microscope and scanning electron microscope observation. Acridine orange staining and tritiated thymidine (3H- TdR) incorporation tests were used to indicate specific killing activity of AS2O3- (HAS-NS)-BDI-l in vitro. Results In AS2O3- (HAS-NS)-BDI-1 groups, we saw two protein bands in SDS-PAGE reduction electrophoresis Albuminutes immuno- nanospheres were rounded with clear green fluorescence by immunofluorescence test. Under microscope, we observed that BIU-87 cells were covered with the AS2O3-(HAS-NS)-BDI-l and that AS2O3-(HAS-NS)-BDI-l moved with the BIU-87 cells. The albuminutes immuno-nanospheres were tightly junctioned with the BIU-87 cells. Specific killing activity of AS2O3- (HAS-NS)-BDI-1 on bladder tumor cells was observed by acridine orange staining and 3H-TdR incorporation assays. Conclusions AS2O3-(HAS-NS)-BDI- 1 might bind specifically against BIU-87 cells, thus leading to high activity of killing bladder tumor cells. Chin Med J 2005; 118(1):50-55

Background Recent studies have revealed the important role of free radicals in renal damage induced by high-energy shock waves (HESW). This study aimed at investigating the effects of Astragalus membranaceus, a traditional Chinese medicinal herb, on free radical-mediated HESW-induced damage to renal tubules in a live rabbit model. Methods Forty-five healthy male New Zealand white rabbits were randomly divided into three groups: control group (n=15), sham group (n=15), and herb-treated group (n=15). Three days prior to HESW application, the controls received verapamil (0.4 mg/ kg), the shams received physiological saline (20 ml), and the herb-treated animals received Astragalus membranaceus (2. 4 g/ kg) intravenously. HESW (1500 shocks, 18kV) was applied to the right kidneys of all anesthetized rabbits. We measured superoxide dismutase(SOD) and malondialdehyde (MDA) levels before and after shock treatment in blood and kidney homogenates. Histopathological changes were also observed. Results MDA levels increased and SOD activity decreased significantly in the sham group (P <0. 05 for both) after shock treatment. MDA levels showed a much less increase in the controls (P<0. 05) and did not increase to statistically significant levels in the group receiving Astragalus membranaceus (P> 0. 05) . SOD values were significantly higher in the controls than in the shams (P <0. 05) . By contrast, SOD levels reoovered rapidly in the rabbits receiving Astragalus membranaceus, reaching a nadir within 24 hours, and returning to baseline more quickly than in control and sham rabbits (P <0. 05) . Histopathological examinations showed that renal tubular damage in the controls was less severe than in the shams, while damage in the Astragalus membranaceus group was even more mild, with rapid recovery in conparison with the controls. Conclusion This study provides preliminary evidence indicating that Astragalus membranaceus has strong protective effects on free radical-mediated renal tubular damage induced by HESW and that these effects are superior to the effects of verapamil. Chin Med J 2005; 118(1):43-49

Background Velvet antler polypeptides (VAPs), which are derived from the antler velvets, have been reported to maintain survival and promote growth and differentiation of neural cells and, especially the development of neural tissues. This study was designed to explore the influence of VAPs on neural stem cells in vitro derived from embryonic rat brain. Methods Neural stem cells derived from E12-14 rat brain were isolated, cultured, and expanded for 7 days until neural stem cell aggregations and neurospheres were generated. The neuroinheres were cultured under the condition of different concentration of VAPs followed by immunocytochemistry to detect the differentiation of neural stem cells. Results VAPs could remarkablely promote differentiation of neural stem cells and most neural stem cells were induced to differentiate towards the direction of neurons under certain concentration of VAPs Conclusion Neural stem cells can be successfully induced into neurons by VAPs in vitro, which could provide a basis for regeneration of the nervous system. Chin Med J 2005; 118(1): 38-42

Background Adult chronic idiopathic thrombocytopenic purpura (ITP) is a common hematologic disease characterized by persistent thrombocytopenia. So far, there were only a few reports on adult Chinese patients with chronic ITP. This study aimed at defining the treatment outcome and prognostic factors for chronic ITP based on a large cohort of Chinese patients followed up for over 25 years at a single center. Methods The medical records of 1791 patients aged 14 years or older who were diagnosed as having chronic ITP at our hospital from 1974 to 1999 were retrospectively analyzed. Results The female-to-male ratio was 21:, with a median age of 34 years (ranging from 14 to 80 years), median platelet count of 38: 109/L [range (1 - 99): 109/L], and median follow-up of 36 months (range 1 -220 months). Steroids were used in 689 patients, among them 209 (30. 3 %) achieved conplete remission(CR) . A splenectomy was performed in 124 patients, and response to steriod pre-splenectomy was not available in 14 patients. The CR rate after a splenectomy was lower in steroid nonreponders (29 of 90, 32. 2 %) than in those who relspsed after successful steroid treatment (12 of 20, 60. 0 %) (P <0. 05) . In comparison with patients negative for antinuclear antibody (ANA), those who were ANA positive had similar responses to steroids, but a significantly shorter remission period after a splenectomy (P <0. 01). Conclusions Adult Chinese chronic ITP patients can have long-term remission after steroid therapy and splenectomies. Primary steroid refractoriness is a prognostic factor predicting poor subsequent response to a splenectomy. Chin Med J 2005; 118(1):34-37

Background The laparoscopic radical cystectomy (LRC) with orthotopic ileal neobladder is now applied to treat invasive bladder cancer, however, it has not been well codified and illustrated We describe in this paper a technique step by step that we have developed in 33 patients and achieved excellent results. Methods The surgical procedure can be divided into eight steps: laparoscopic pelvic lymphadenectomy and mobilization of the distal ureters; exposing Denonvillier's space and the posterior aspect of prostate; exposing retropubic space and anterior surface of the bladder; dividing the lateral pedicles of the bladder and the prostate; dividing the apex of the prostate; extracorporeal formation of the ileal pouch; extracorporeal implantation of the ureters; and 1aparoscopic urethra-neobladder anastomosis. This operation was performed in 33 patients, 29 males and 4 females, with muscle invasive bladder cancer between December 2002 and September 2004. Results The operating time was 5.5- 8. 5 hours with an average of 6. 5 hours; the estimated blood loss was 200 - 1000 ml with an average of 460 ml. The surgical margins of the bladder specimen were negative in all patients. There was no evidence of local recurrence at follow-up of 1 - 21 months in all the patients. However lymph node metastases were found in one case at 9 months postoperatively. Most of patients achieved urine control 1 to 3 months after surgery. The daytime continence rate was 94%(31 cases) and nighttime continence rate was 88 %(29 cases) . Urodynamic evaluation was performed between 3 and 6 months postoperatively for all cases. The mean value of neobladder capacity was (296 ±37) ml. The mean value of maximum flow rate was (18. 7+7. 1) ml/ s. The mean residual urine volume was (32 ±19) ml. In all cases, excretory urography at 1 to 2 months postoperatively demonstrated slightly dilated upper urinary tracts without ureteral obstruction, which resolved at follow up. Cystography showed neobladders being similar in shapes to normal. Two small ureteral nipples with intermittently efflux of urine were observed at cystoscopy in most patients. Postoperative conplications occurred in 6 of 33 patients (18 %), including pouch leakage in 2 cases, pelvic infection in 1, partial small bowel obstruction in 2 and neobladdervaginal fistula in 1. Conclusions The LRC with orthotopic ileal neobladder is a feasible option for bladder cancer when radical cystectomy is indicated. The extracorporeal formation of the ileal pouch and ureteral implantation through a small lower midline incision can simplify the complexity of the procedures, shorten the duration of surgery and reduce the medical expenses. Chin Med J 2005; 118(1):27-33

Background Pulmonary artery smooth muscle cell (PA SMC) proliferation plays an inportant role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cy to skeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pul monary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligo deoxy nucleotide s (ODNs) to selectively suppress the expression of FAK protein. Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expession of FAK, Jun NH2-terminal kinase(JNK), cyclin-dependent kinase 2 (CDK2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining. Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression up regulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense- FAK ODNs group. The level of cell apoptosis in antisense-FAK group was higher than in the mismatch sense group and the latter was higher than sense-FAK group. In addition, the sense-FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense-FAK ODNs group was weakly stained. Conclusions The results suggest that FAK relates to the proliferation of HPASMCs. Antisense-FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis. It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase-3 inhibits HPASMCs apoptosis. Chin Med J 2005; 118(1):20-26

Background Hypoxic pulmonary hypertension (HPH) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs) . The mechanism of hypoxic pulmonary hypertension has not yet been fully elucidated. The mitochondrial ATP-sensitive K+ channel(Mito KATP) is extremely sensitive to hypoxia, and is a decisive factor in the control of mitochondrial membrane potential(Δψm) . This study investigated the changes of cell membrane potential and Kv channel in cultured human pulmonary artery smooth muscle cell (hPASMC) exposed to 24 hour hypoxia, and explored the role of Mito KATP and Δψm in this condition. Methods Fresh human lung tissues were obtained from the patients undergoing a chest operation. hPASMCs were isolated, cultured, and divided into 6 groups: ①control group, cultured under normoxia; ②diazoxide groip, cultured in normoxia with diazoxide, an opener of Mito KATP; ③5-HD group, cultured in normoxia with sodium 5- hydro xydecanoate (5-HD), an antagonist of Mito KATP; ④24 hour hypoxia group;⑤24 hour hypoxia + diazoxide group; and ⑥ 24 hour hypoxia + 5HD group. Whole-cell patch-clamp technique was used to trace the cell membrane K+ currents. The expressions of cell membrane Kvl. 5 mRNA and protein were determined by RT-PCR and Western blot technique, respectively. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. Results After exposure to diazoxide for 24 hours, the intensity of R-123 fluorescence in normoxic hPASMCs was significantly increased co mpared with control group (P<0.05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5-HD for 24 hours. Twenty-four hour hypoxia or 24 hour hypoxia + diazoxide could markedly increase the intensity of R-123 fluorescence in hPASMC and the changes were more significant in 24 hour hypoxia + diazoxide group than in 24 hour hypoxia group (P < 0.05) although 5-HD could partly weaken the effect of 24 hour hypoxia on the intensity of R-123 fluorescence. After exposure to diazoxide for 24 hours, the cell membrane K+ currents and the expression of cell membrane Kvl. 5 mRNA and protein in normoxic hPASMCs were significantly decreased conpared with control group (P <0. 05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5-HD for 24 hours. Also, 24 hour hypoxia or 24 hour hypoxia + diazoxide decreased the cell membrane K+ currents and the expression of Kvl. 5 mRNA and protein (P <0. 05) but the changes were more significant in 24 hour-hypoxia + diazoxide group than in 24 hour hypoxia group (P <0. 05). Again, 5-HD could partly weaken the inhibitory effect of 24 hour hypoxia on the cell membrane K+ currents and the expression of Kvl. 5 mRNA or protein (P <0. 05). Conclusions The opening of Mito KATP followed by the alterations in the expression of cell membrane Kvl. 5 mRNA and protein leading to change in the cell membrane potential of hypoxic hPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension. Chin Med J 2005; 118(1): 12-19