2003年 · 第116卷第12期

Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells. Methods Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2. 7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer(MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient. Results One hour after apoptosis induction significant cell fractions were positively stained for ssDNA(33%with MiaPaCa-2 cells, 35%with Hep-G2 cells, 56%with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2. 7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90%positively stained necrotic cells by using Apo2. 7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type. Conclusions Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2. 7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis. Chin Med J 2003; 116(12): 1923-1929

Objective To investigate the insulin-like growth factor- I (IGF- I) gene and polypeptide expression in cultured rat osteoblast (ROB) and the role of IGF- I in mediating the cell-to-cell communication by mimicking the pharmacokinetics of parathyroid hormone (PTH). Methods The ROB was cultured with three kinds of treatment: (1) Control (Ctr), the cells were cultured without PTH during the first 6 hours and the subsequent 42 hours in a 48-hour cycle; (2)Intermittent exposure to PTH (Itm), the cells were cultured with PTH during the first 6 hours, but without PTH in the subsequent 42 hours; and (3) Continuous exposure to PTH (Ctu), the cells were cultured with PTH during the first 6 hours and the subsequent 42 hours. Results The bone-forming activities of ROB were increased in Itm and inhibited in Ctu. The IGF- I mRNA content in Itm cells was elevated only during the first 6 hours and that in Ctu cells was elevated at any time during an incubation cycle. The free IGF- I concentration in the medium of Itm cells was generally higher and that of the Ctu cells was generally lower compared with those of the Ctr cells. The IGF- I antibody significantly reduced the alkaline phosphatase activity within the cells of Ctr and Itm. Conclusions PTH rapidly and constantly stimulates the IGF- I gene transcription of osteoblast. There was an obvious discrepancy between the IGF- I mRNA content within the osteoblast and the free IGF- I level around the osteoblast in either mode of PTH action. The IGF- I might be important for osteoblast-osteoblast communication and bone-forming activity, not only in intermittent PTH administration, but also in the physiological functioning of osteoblasts. Chin Med J 2003; 116(12): 1916-1922

Objective To study the dose- and time- dependent protective effects and the synergistic effects of nimodipine (NMDP) and fructose-1, 6-diphosphate (FDP) against cerebral damage induced by acute carbon monoxide (CO) poisoning in mice. Methods Male mice were exposed to CO 170 mL/kg, i. p. After CO intraperitonealy exposure, mortality of mice, change in memory function estimated by passive avoidance test, the pathomorphologic observation of brain tissue slices, as well as changes of activities of monoamine oxidase (MAO)-B and Ca 2+-Mg 2+-ATPase in cerebral tissue were studied. In dose-dependent protective effect study, NMDP (10. 6, 5. 3, 2. 7 mg/kg) and FDP (2. 6, 1. 3, 0. 67 g/kg) was injected ip, respectively 15 min after CO exposure. To study the time-effect relationship of drugs,NMDP (5. 3 mg/kg) and FDP (1. 3 g/kg) were administered ip respectively 15 minutes, 45 minutes and 120 minutes after CO exposure. The combination of NMDP (2. 7 mg/kg) and FDP (0. 67 g/kg)was administered ip15 minutes, 45 min and 120 minutes after CO exposure to study the synergism of the two drugs. Results Either NMDP (10. 6, 5. 3 mg/kg) or FDP (2. 6, 1.3 g/kg) administered ip within 15minutes after CO exposure significantly decreased the impairment of memory function and mortality rate induced by CO, inhibited the decrease of Ca 2+-Mg 2+-ATPase activity, blunted the rising of MAO-B activity and prevented the delayed hippocampal neuronal death in poisoning mice. To our surprise,the combined use of NMDP (2. 7 mg/kg) and FDP (0. 67 g/kg) within 15 minutes after CO exposure had similar effects to that in NMDP (10. 6, 5. 3 mg/kg) and FDP (2. 6, 1. 3 g/kg). Conclusions These results suggest that the impairment of CO on brain can be attenuated if NMDP or FDP are administered sufficiently and quickly as soon as possible after CO exposure and there exists a synergism of FDP and NMDP against CO poisoning damage. Chin Med J 2003; 116(12): 1911-1915

Objective To investigate the mechanism (s) of penicillins allergic reaction. Methods The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl(AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test. Results Nineteen of 32 patients (59. 4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34. 38%), followed by BPO-IgE (31. 25%). The positive rate of FLUO-IgE was the lowest (15. 63%). Of the total patient group, 53. 13%were positive to one or more minor antigenic determinants, while 37.5%(12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63. 16%) was significantly higher than observed in the anaphylactic shock group(38.5%, P<0. 05). Conclusions The minor antigenic determinant was important in allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule. Chin Med J 2003; 116(12): 1904-1910

Objective To elucidate the inhibitory effects of recombinant Chinese scorpion neurotoxin BmK IM on seizures induced by pentylenetetrazol (PTZ) and the possible mechanism. Methods After purifying recombinant BmK IM from an E. coli cell line, its toxicity (both LD50 and minimum lethal dose) on rats was determined. BmK IM was then microinjected into the CA3 region of the right hippocampus and its ability to inhibit the effects of an intraperitoneal injection of PTZ was assessed. The effects of BmK IM on the electrophysiological properties of isolated CA3 pyramidal neurons were then studied using whole-cell patch clamp techniques. Results BmK IM can significantly prolong the latent period of epileptic seizures, decrease the degree of seizures, and decrease the frequency of epileptiform discharges induced by PTZ. At the same time, 24h after injection of BmK IM into the hippocampal tissue, BmK IM significantly reduces the concentration of the neurotransmitter glutamate and alleviates PTZ-induced lesions in the hippocampus. Whole-cell patch clamp recordings indicate that BmK IM inhibits lNa of rat hippocampal neurons in a dose-dependent manner. BmK IM significantly shifts the activation curve of INa in a positive direction, indicating that BmK IM enhances the threshold potential of INa. Conclusions BmK IM has significant anti-epileptic properties, and may prove useful as a drug in the therapy of epilepsy. The inhibitory effects of BmK IM on seizures caused by pentylenetetrazol might depend on reductions in the release of presynaptic glutamate via the blocking of Na+ channels. Chin Med J 2003; 116(12): 1898-1903

Objective The goal of this study is to investigate the inappropriate activation of Wnt pathway in the hepatocarcinogenesis. Methods We analyzed the alterations of three key components of Wnt pathway, β-catenin, glycogen synthase kinase 3β (GSK-3β) and T cell factor 4 (Tcf-4), in 34 samples of hepatocellular carcinoma(HCC) and paracancerous normal liver by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Results We found 61. 8%(21/34) of all the HCCs examined showed an abnormal β-catenin protein accumulation in the cytoplasm or nuclei. RT-PCR-SSCP and direct sequencing showed that β-catenin exon 3 mutations existed in 44. 1%(15/34) of the HCCs. No mutations of GSK-3β or Tcf-4 were detected in HCCs. Moreover, mRNA of β-catenin and Tcf-4 but not GSK-3β was found to be over expressed in HCCs. On analyzing the relationship between alterations of β-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that the mutations of β-catenin as well as over expression of β-catenin or Tcf-4 gene were independently correlated with C-myc gene over expression in HCCs. Conclusions Our present findings strongly suggest mutations of β-catenin as well as over expression of β-catenin and Tcf-4 gene activate the Wnt pathway in HCC independently with the target gene most likely to be C-myc. Chin Med J 2003; 116(12): 1885-1892

Objective To investigate the anti-idiotypic effect induced by a monoclonal antibody. Methods A conventional fusion method was used to obtain hybridoma cells produing monoclonal antibody, which were detected by flow cytometry. ELISA were used to detect the humoral response induced by the antibody in mice. Cytotoxic and proliferation experiments were used to detect the cellular response induced by the antibody in mice. Results CS20 is a MUC-1 specific monoclonal antibody that strongly reacts with MUC-1 antigen expressed on the cell surface of breast cancer cells. The antibody could not kill tumor cells directly through complement-dependant cytotoxicity or antibody-dependant cell-mediated cytotoxicity. However, after 6 administrations of mAb CS20-KLH (keyhole limpet hemocyanin) conjugated to BALB/c mice (n=5) at a dose of 50 μg/mouse, anti-idiotypic antibodies and anti-anti-idiotypic antibodies were induced. T cells derived from CS20-KLH-immunized mice responded to mAb CS20,indicating the existence of idiotype-specific T cells. Conclusion These data indicated the possibility of using MUC-1 specific antibody for active immunotherapy of breast cancer. Chin Med J 2003; 116(12): 1879-1884

Objective To investigate the link between the polymorphism of -109 and Glu237 in the high-affinity IgE receptor β (FceR I β) gene and susceptibilty to allergic asthma in a Chinese population. Method Blood samples from 216 allergic asthma patients and 198 age- and sex-matched controls were studied. A -109C/T and a coding variant Glu237Gly in FceR I β were detected with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Results The genotype frequencies were 0. 403 for - 109T/T, 0. 491 for - 109T/C and 0. 106 for-109C/C in allergic asthma in a Chinese population. No significant difference in the distribution of-109C/T polymorphism was found between allergic asthma subjects and healthy controls, however, homozygosity for the - 109T allele was associated with increased total plasma IgE levels in subjects with allergic asthma (F=4. 020, P <0. 05). The allele frequency of Gly237 in the patients and control was 0. 236 and 0. 136 respectively. There was a significant association between the Gly/Gly genotype and allergic asthma. Among allergic asthma patients Gly237 was significantly associated with high IgE levels. Conclusions These results suggest that the Gly237 variant of the FceR I β gene is involved in the development of allergic asthma. The - 109C/T and Glu237Gly polymorphisms are two of the genetic factor identified thus far, which affect total plasma IgE levels of allergic asthma patients in a Chinese population. Chin Med J 2003; 116(12): 1875-1878

Objective To investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization. Methods pBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe I and Sal I, by PCR reaction, by sequence, and then by alignment of PCR products with the geneBank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75%AgNO3 and 25%KNO3 cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs. Results pBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40. 2%, and 42. 8%respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity. Conclusions The plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition. Chin Med J 2003; 116(12): 1869-1874

Objective To delineate the influence of transcutaneous electrical nerve stimulation (TENS) on heat pain threshold and vibration threshold in human. Methods Twenty healthy, young subjects, aged from 20 to 39, participated in the study. They were randomly allocated into either TENS or placebo group. Thirty minutes of conventional TENS (200 μs pulses at 100 Hz and 2-3 times sensory threshold) or placebo stimulation was applied to the acupuncture points (LI4) on each subject's left hand. Heat pain and vibration thresholds were measured using Medoc TSA-2001 and VSA-3000 respectively on the thenar eminence of each subject's left hand. These assessments were done at 30, 20 and 10 minutes before and 0, 10, 20,30, 40 and 50 minutes after 30 minutes of TENS or placebo treatment. Results This study showed a significant increase in heat pain threshold by 0. 81℃ and 1. 54℃respectively at 0 (P=0.002) and 20 minutes (P=0.004) after 30 minutes of TENS application to the LI4 acupoint of young healthy subjects, compared with placebo stimulation. Interestingly, no significant difference in vibration threshold was found between the TENS and placebo groups. Conclusion The effect of TENS on the acupoint is to reduce pain but not tactile (specifically vibration) sensibility. Chin Med J 2003; 116(12): 1864-1868

Objective To investigate feasible treatment methods for plantar ulcers in leprosy patients according to the agreement between the Ministry of Health (MOH) of China and the Leprosy Mission International(LMI). Methods A total of 2599 complicated foot ulcers in 1804 leprosy cases underwent surgic treatment. Plastic fixation and supports were used, dressings were changed regularly, and protective footwear and modified insoles were provided. Results Of the 2599 foot ulcers 1446 (55. 64%) healed. The cure rate of the patients treated in leprosy hospitals was 71. 31 %, with 219 (15. 15%) recurrences of foot ulcers. The recurrence rate of those who lived at home was 18. 35%. Conclusions Comprehensive treatment of foot ulcers has a high cure rate and a low recurrence rate. Reduction of workload, avoidance of long distance walking, intensification of education on foot self-care and provision of financial support are the main measures for preventing a recurrence of foot ulcers. Chin Med J 2003; 116(12): 1946-1948

Objective To report the surgical techniques and results of an 8-year follow-up study of 42 patients with a modified orthotopic ileal neobladder restoring normal anatomical relationship. Methods Total cystoprostatectomy was performed extraperitoneally. A 45 - 50 cm segment of the ileal loop was isolated, detubularized, and reconfigured into an "M"-shape to form a pouch. Bilateral ureters were implanted by inserting 1 cm distal segment into the pouch. The bottom of pouch was opened and anastomosed with the urethra. Results Forty-two patients were followed up for 6 to 96 months, 90. 5%of whom were continent in the daytime, and 85. 7%at night. Two patients had a difficulty in urination. The average volume of the pouch was (361 ±48) ml at 12 months postoperation. Urodynamic examination showed the average peak voiding pressure was (86. 8 ±21. 4) cmH2O. The average maximum flow rate (Qmax) was(18. 4 ±6. 1) ml/s. No remarkable ureter reflux and obstruction were found. No patient was detected to have urethral carcinoma. Conclusions Extraperitoneal cystectomy can avoid the tumor contamination of the abdomen and intestinal interference of the operative field. The ureter-inserting implantation technique is a simple anti¬reflux anastomosis method with less ureter stenosis rate. Isolating the neobladder and ureters from the peritoneal cavity can reduce the postoperative complications, such as adhesive ileus, internal hernia, and urine leakage into the peritoneal cavity. The neobladder is similar to the original bladder in position, volume, shape and anti-reflux ureter connection. Chin Med J 2003; 116(12): 1943-1945

Objective To discuss the relationship between the onset of peptic ulcers (PU) and meteorological factors (MF). Methods In reviewing records from 17 hospitals in the city of Nanning from 1992 to 1997, we found 24, 252 cases of PU in 104, 121 samples of gastroscopic examinations. We then calculated the detectable rate of PU (DRPU) during each season every five days (FD) and made a correlated analysis with the seasonal MF during the same period in Nanning. Finally, we made a multiple regressive correlated analysis of DRPU and the 5MF for the same period of the year. A forecast model based on the MF of the previous FD was established. The real value and the forecast value was being tested and verified. Results From 1992 to 1997, the DRPU is: winter and spring>summer and autumn (P <0. 005). There is a close relationship between the DRPU and the average temperature (AT), the average highest temperature (AHT), the average lowest temperature (ALT), average air pressure (AAP)and the average dew point temperature (ADT) of the five days of the same period of the year (the correlated coefficients are -0. 5348, -05167, -0. 5384, 0. 4579 and -0. 4936, respectively), with P <0. 01. The AT, AHT, ALT, AAP and ADT of the previous FD are of great value in forecasting the onset of PU, with its real value and forecast value corresponding to 66. 6%. Conclusions There exists a close relationship between DRPU and the AT, AHT, ALT, AAP and ADT of the FD for the same period. A mid-term medical meteorological forecast of the onset of PU can be made more accurately and reliably according to the close relation betweenthe DRPU and some MF of the previous FD. Chin Med J 2003; 116(12): 1940-1942

Objective To investigate hypoxia-inducible factor 1α (HIF-1α) protein expression in normal prostates(NP), benign prostatic glandular hyperplasia (BPH), and prostate adenocarcinoma (Pca). Methods HIF-1α protein expression was determined by immunohistochemistry in formalin-fixed and paraffin-embedded specimens obtained from 13 cases of NP, 28 cases of BPH, and 34 cases of Pca. In cases of Pca, the relationship between HIF-1α protein expression and certain clinicopathological factors, such as clinicopathologic stage and Gleason score, was evaluated. Results NP manifested no immunoreactivity, whereas Pca and BPH showed significantly increased HIF-1 a protein expression. A significantly higher expression was observed in Pca specimens compared with BPH samples. In Pca, no significant relationship between HIF-1 a protein expression and clinicopathological factors was found. Conclusion Our findings of increased HIF-1 a protein expression in BPH and Pca specimens suggests the potential role of this protein in BPH and Pca. Chin Med J 2003; 116(12): 1936-1939

Objective To investigate the association of fasting and postpiandial triglyceride with carotid intima-media thickness among type 2 diabetic patients in southeast China. Methods A total of 78 type 2 diabetic patients from southeast China were recruited. All the subjects underwent a standardized mixed-meal test (SMMT) and an oral glucose tolerance test. Carotid intima-media thickness (IMT) was measured by color doppler ultrasound examination. Results Postprandial triglyceride (PTG) after SMMT increased significantly in type 2 diabetic patients. The carotid IMT in patients with postprandial hypertriglyceridemia was sinificantly greater than that in patients with postprandial normal-triglyceridemia (0. 90 mm vs 0. 81 mm, P <0. 05), which remained significant after adjustment for fasting TG and HDL-c levels, and the IMT in patients with fasting hypertriglyceridemia only had tendency to increase compared to patients with fasting normal-triglyceridemia (0. 87 mm vs 0. 84 mm, P>0. 05). Conclusion The results suggest that postprandial hypertriglyeridemia may be an independent risk factor of early atherosclerosis in type 2 diabetes. Chin Med J 2003; 116(12): 1933-1935

Objective To observe the clinical effects of lesionectomies combined with bipolar coagulation of the surrounding cortex (BCSC) on symptomatic epilepsy. Methods The effectiveness of lesionectomies combined with BCSC (71 patients) and of lesionectomies alone (78 patients) were compared in follow-up surveys conducted 2-5 years(average of 37 months) post-operation. Results Treatment in the BCSC group was significantly more effective than in the lesionectomy group(P<0.01). Meanwhile, patients in the BCSC group suffered no permanent post-operative complications. Conclusion Lesionectomy combined with BCSC is a new, effective and safe treatment for symptomatic epilepsy. Chin Med J 2003; 116(12): 1930-1932