2004年 · 第117卷第07期

Objective The aim of this review was to assess RNA interference (RNAi) and its possibility as a potential and powerful tool to develop highly specific double-stranded RNA (dsRNA) or small interfering RNA (siRNA) based gene-silencing therapeutics. Data sources The data used in this review were obtained from the current RNAi-related research reports. Study selection dsRNA-mediated RNAi has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides siRNAs trigger gene-specific silencing in mammalian cells has further expanded the utility of RNAi in to the mammalian system. Data extraction The currently published papers reporting the discovery and mechanism of RNAi phenomena and application of RNAi on gene function in mammalian cells were included. Data synthesis Since the recent development of RNAi technology in the mammalian system, investigators have used RNAi to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous siRNA or siRNA expressing vector. The general and sequence-specific inhibitory effects of RNAi that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality. Conclusions It is certain that the ability of RNAi in mammals to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that RNAi might one day be used to treat human diseases. Chin Med J 2004; 117(7):1084-1091

Background Hereditary spastic paraplegia is a clinically and genetically heterogeneous group of neurodegenerative disorders of the motor system, characterized by slowly progressive spasticity and weakness of the lower extremities. This study was conducted to investigate the clinical features of hereditary spastic paraplegia with thin corpus callosum (HSP-TCC). Methods Clinical data from five patients and thirty-five previously published case reports of HSP-TCC were analyzed retrospectively. Results Most patients were adolescents at the onset of the disease, presenting with spastic paraparesis of the lower limbs and mental impairment. Some patients also had other clinical features, including spasticity of the upper limbs, cerebellar ataxia, and sensory disturbances. Cranial MRIs of the five patients revealed an extremely thin corpus callosum, sometimes with widened cerebral sulci and ventricles, as well as with cerebellar and cerebral atrophy. Conclusion The main clinical features of HSP-TCC include slowly progressive spastic paraplegia, mental impairment during the second decade of life, and an extremely thin corpus callosum as shown on cranial MRIs. Chin Med J 2004; 117(7):1002-1005

Background Non-penetrating trabecular surgery is a new filtrating surgery without opening in ternal trabecular structures. This study was to estimate the overall efficacy of non-penetrating trabecular surgery for open angle glaucoma. Methods The published articles selected for this study were obtained by a computerised Medline and China Biological Medicine Disk search of the literature and a manual search of the bibliographies of relevant articles. Articles meeting the inclusion criteria were reviewed systematically, and the reported data were aggregated using the statistical techniques of meta-analysis. Results A total of 37 articles were included in the meta-analysis. The pooled complete success rates of non-penetrating trabecular surgery with different techniques were: deep sclerectomy single, 69. 7% (95%CI: 58. 5% - 81. 0%); deep sclerectomy with collagen implant, 59. 4% (95% CI: 47. 0% - 71. 8%); deep sclerectomy with reticulated hyaluronic acid implant, 71. 1% (95% CI: 56. 8% - 85. 3 %); and viscocanalostomy, 72. 0% (95% CI: 57. 6% - 86. 4%). The overall weighted complete success rate of non-penetrating trabecular surgery was 67. 8% (95% CI: 61. 4% - 74. 3%). Conclusions Non-penetrating trabecular surgery is the best available therapy method for medically uncontrolled open angle glaucoma with a complete success rate of over 60%. But the different techniques cannot belie the complete success rate of non-penetrating trabecular surgery. Chin Med J 2004; 117(7):1006-1010

Background Functional neuroimaging has been used in neurolinguistic research on normal subjects and on patients with brain damage. This study was designed to investigate the differences of the neural basis underlying language processing between normal subjects and aphasics. Methods Functional magnetic resonance imaging (fMRI) was used to map the language network in 6 normal subjects and 3 patients with aphasia who were in the stage of recovery from acute stroke. The participants performed a word generation task during multi-slice functional scanning for the measurement of signal change associated with regional neural activity induced by the task. Results In normal subjects, a distributed language network was activated. Activations were present in the frontal, temporal, parietal and occipital regions. In the patient group, however, no activation was detected in the left inferior frontal gyrus whether the patient had a lesion in the left frontal lobe or not. Two patients showed activations in some right hemisphere regions where no activation appeared in normal subjects. Conclusions fMRI with word generation task is feasible for evaluating language function in aphasic patients. Remote effect of focal lesion and functional redistribution or reorganisation can be found in aphasic patients. Chin Med J 2004; 117(7):1011-1016

Background The aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation. Methods Full-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed. Results The transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA. Conclusions Activation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor. Chin Med J 2004; 117(7):1017-1022

Background Hypoxia-inducible factor-1α (HIF-1α) is one of the pivotal mediators in the response of lungs to decreased oxygen availability, and increasingly has been implicated in the pathogenesis of pulmonary hypertension. Vascular endothelial growth factor (VEGF), a downstream target gene of HIF-1α, plays an important role in the pathogenesis of hypoxic pulmonary hypertension and hypoxic pulmonary artery remodelling. In this study, we investigated the dynamic expression of HIF-1α and VEGF in pulmonary artery of rats with hypoxia-induced pulmonary hypertension. Methods Forty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were estimated. Lungs were inflated and fixed for in situ hybridisation and immunohistochemistry. Results mPAP values were significantly higher than the control values after 7days of hypoxia [(18. 4 ± 0. 4) mmHg, P <0. 05]. RVHI developed significantly after 14 days of hypoxia. Expression of HIF-1α protein increased in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterial tunica media, HIF-1α protein was markedly increased by day 3 (0. 20 ± 0. 02, P<0. 05), reached the peak by day 7, then declined after day 14 of hypoxia. HIF-1α mRNA increased significantly after day 14 of hypoxia (0. 20 ± 0. 02, P<0. 05). VEGF protein began to increase markedly after day 7 of hypoxia, reaching its peak around day 14 of hypoxia (0. 15±0. 02, P<0. 05). VEGF mRNA began to increase after day 7 of hypoxia, then remained more or less stable from day 7 onwards. VEGF mRNA is located mainly in tunica intima and tunica media, whereas VEGF protein is located predominantly in tunica intima. Linear analysis showed that HIF-1α mRNA, VEGF and mPAP were correlated with hypoxic pulmonary artery remodelling. HIF-1α mRNA was positively correlated with VEGF mRNA and protein (P<0. 01). Conclusion HIF-1α and VEGF are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats. Chin Med J 2004; 117(7):1023-1028

Background Raloxifene has been approved for prevention and treatment of postmenopausal osteoporosis in Caucasian women. It also has some positive effects on serum lipids in Caucasians. The objective of this study was to determine the effect of raloxifene hydrochloride on lumbar spine and total hip bone mineral density (BMD), bone metabolism, and serum lipids in Chinese postmenopausal women with osteoporosis. Methods This was a multi-center, randomized, double-blind, placebo-controlled clinical trial in which 204 postmenopausal Chinese women with osteoporosis were assigned to receive raloxifene (60 mg) or placebo treatment daily for 12 months. BMD, serum bone metabolism markers, and serum lipids were measured before and after drug administration. BMD was measured by Dual-Energy X-Ray Absorptiometry (DEXA) and bone metabolism markers were analyzed by one-step enzyme-linked immunosorbent assay. Serum lipids were measured by enzymatic analysis. Results At the end of the 12-month study, lumbar spine BMD increased in both groups with a mean increase of (3. 3±4. 8) % in the raloxifene group and (1. 0±4. 9) % in the placebo group (P<0. 001). There was a mean increase in total hip BMD of (1. 4 ±4. 8) % in the raloxifene group and a mean decrease of (0. 9±5. 0) % in the placebo group (P<0. 001). No subject in the raloxifene group had a new vertebral fracture and 5 placebo subjects had new fractures (P > 0. 05). In the raloxifene group, the median decreases in the biochemical markers of bone metabolism serum osteocalcin and C-telopeptide were 41. 7% and 61. 5%, respectively. These changes were statistically significant compared with those in the placebo group (10. 6% and 35. 6%, P < 0. 001, respectively). Both total cholesterol and low-density lipoprotein cholesterol decreased significantly in the raloxifene group compared with those in the placebo group (P<0. 001, respectively) and there was no significant effect of raloxifene on high-density lipoprotein cholesterol and triglycerides compared with placebo. Conclusions Raloxifene 60 mg/d for 12 months significantly increases lumbar spine and total hip BMD, significantly decreases bone turnover, and has favourable effects on serum lipids in Chinese postmenopausal women with osteoporosis. Chin Med J 2004; 117(7):1029-1035

Background Activating on mammalian and human body LDR is thought to induce adaptive response, enhance immune function and increase anti-tumor ability. This study was designed to assess the effect of low-dose radiation on tumor growth and on erythrocyte immune function and superoxide dismutase (SOD) activity in tumor-bearing mice. Methods Male Kunming mice were subcutaneously implanted with S180 sarcoma cells in the right inguen to create an experimental in situ animal model. Six hours before implantation, the mice were given 75 mGy X-ray radiation, over the body. Tumor size was observed 5 days later while tumor volume was calculated every other day, allowing for the creation of a graph depicting tumor growth. Fifteen days after implantation, the mice were killed to measure tumor weight and observe the necrotic areas and the location of tumor-infiltrating lymphocytes (TILs). Erythrocyte immune function and SOD activity were also determined. Results Mice pre-exposed to low-dose radiation had a lower tumor formation rate than did those receiving no radiation (P<0. 05). Tumor growth was significantly lower in the mice pre-exposed to low-dose radiation; after 15 days, the average tumor weight in the mice pre-exposed to low-dose radiation was also lower (P<0. 05). Areas of tumor necrosis and infiltration of TILs were larger in the low-dose radiation group than in the non-radiation group. Erythrocyte immune function and SOD activity were higher in the low-dose radiation group than in the non-radiation group (P<0. 05). Conclusion Low-dose radiation can markedly increase the anti-tumor ability of an organism and improve erythrocyte immune function and red blood cell SOD activity as well, suggesting that low-dose radiation might be useful in the clinical treatment of cancer. Chin Med J 2004; 117(7):1036-1039

Background The changes in matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions were examined in the kidneys of diabetic rats to investigate the degradative pathway of collagen type Ⅳ (C-Ⅳ) and the protective effects of pioglitazone on an experimental model of diabetic nephropathy. Methods In 54 SD rats used in our study, 18 served as normal controls. Diabetes mellitus was induced in 36 age- and weight-matched rats by intraperitoneal injection of streptozotocin (70 mg/kg); 18 of the diabetic rats were allocated at random to receive pioglitazone (20 mg • kg-1 • d-1) in their drinking water and 18 served as diabetic controls. Rats were killed after 2, 4, or 8 weeks of treatment. Kidneys were examined pathomorphologically and the expressions of MMP-2, MMP-9, and C-Ⅳ were analyzed by immunohistochemistry, and the results were quantified by image analysis techniques. Results Diabetes mellitus was associated with a decrease in the expression of MMP-2 in the glomeruli (P<0. 05, vs control). By contrast, MMP-2 expression in the interstitium increased, but not significantly (P>0. 05, vs control). The expression of MMP-9 did not show any change when comparing the three groups (P>0. 05, vs control). STZ-diabetic rats were also associated with an increase in the expression of C-Ⅳ in the glomeruli and the interstitium (P<0. 05, vs control). All diabetes-associated changes in MMP-2 expression were attenuated by pioglitazone treatment in association with reduced C-Ⅳaccumulation. Conclusions These results indicate that a decrease in MMP-2 expression in the glomeruli of diabetic rats may lead to impairment of C-Ⅳ degradation and contribute to the matrix accumulation in diabetic nephropathy. Pioglitazone treatment, which can attenuate the decrease of glomerular MMP-2 and the increase of C-Ⅳ degradation, has curative effects on diabetic nephropathy. Chin Med J 2004; 117(7):1040-1044

Background In obscure gastrointestinal (GI) bleeding, it is often difficult to detect the bleeding sites located in the small bowel with conventional radiological, scintigraphic or angiographic techniques. Push enteroscopy and capsule endoscopy are currently considered to be the most effective diagnostic procedures. The aim of this study was to compare the detection rates between capsule endoscopy and push enteroscopy. Methods From May 2002 through January 2003, we prospectively examined by capsule endoscopy 39 patients with suspected small bowel diseases, in particular GI bleeding of unknown origin in Renji Hospital. Among them, 32 complained of obscure recurrent GI bleeding. Between January 1993 and October 1996, we used push enteroscopy on 36 patients who suffered from unexplained GI bleeding. All patients had prior normal results on gastroscopy, colonoscopy, small bowel barium radiography, scintigraphy and/or angiography. Results M2A capsule endoscopy disclosed abnormal small bowel findings in 26 (82%) out of 32 patients. Twenty-one of them had significant pathological findings explaining their clinical disorders. Diagnostic yield was therefore 66% (21 of 32 patients). Definite bleeding sites diagnosed by capsule endoscopy in 21 patients included angiodysplasia (8), inflammatory small-bowel (5), small-bowel polyps (4), gastrointestinal stromal tumour (2), carcinoid tumour and lipoma (1), and hemorrhagic gastritis (1). Push enteroscopy detected the definite sources of bleeding in 9 (25%) of the 36 patients. Patients with definite bleeding sources included angiodysplasias (2), leiomyosarcoma (2), leiomyoma (1), lymphoma (1), Crohn's disease (1), small-bowel polyps (1) and adenocarcinoma of ampulla (1). Suspected bleeding sources were shown by push enteroscopy in two additional patients (6%), and in other five patients (16%) by capsule endoscopy. Conclusions The present study of patients with obscure GI bleeding showed that capsule endoscopy significantly superior to push enteroscopy in detecting GI bleeding (P<0. 001). Capsule endoscopy is safe and painless, and should become the initial diagnostic choice for patients with obscure GI bleeding. Chin Med J 2004; 117(7):1045-1049

Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method. Methods Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified. Results Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22. 8% (16/70) were recovered after slow freezing (P<0. 01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers. Conclusions Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation. Chin Med J 2004; 117(7):1050-1055