2002年 · 第115卷第08期

Objective To investigate apoptosis in XG-7, a human myeloma cell line, induced by IL-6 deprivation and the function of three anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL, Mcl-1) in the apoptotic process. Methods Apoptosis in XG-7 myeloma cells induced by IL-6 withdrawal was determined by flow cytometry with propidium iodide (PI) nuclear staining. Expressions of three Bcl-2 proteins in XG-7 cells were monitored by immunoblotting assay. Results In the absence of IL-6 for a certain time, a significant percentage of apoptiotic XG-7 cells can be observed, as well as down-regulated expression of one of the three anti-apoptotic proteins (Mcl-1) in XG-7 cells. IL-6 re-stimulation in XG-7 cells following cytokine removal up-regulated the expression of Mcl-1 and inhibited cell apoptosis. Conclusion Mcl-1, instead of Bcl-2 and Bcl-xL, plays an important role in IL-6 deprivation induced apoptosis in XG-7 human myeloma cells. Chin Med J 2002; 115(8): 1241-1243

Objectives To formulate an equation for fine mapping of disease loci under complex conditions and determine the marker-disease distance in a specific case using this equation. Methods Lewontin ' s linkage disequilibrium (LD) measure D' was used to formulate an equation for mapping disease genes in the presence of phenocopies, locus heterogeneity, gene-gene and gene-environment interactions, incomplete penetrance, uncertain liability and threshold, incomplete initial LD,natural selection, recurrent mutation, high disease allele frequency and unknown mode of inheritance. This equation was then used to determine the distance between a marker (ε4 within the apolipoprotein E gene,APOE) and Alzheimer's disease (AD) loci using published data. Results An equation was formulated for mapping disease genes under the above conditions. If these conditions are present but ignored, then recombination fraction 0 between marker and disease loci will be either overestimated or estimated with little bias. Therefore, an upper limit of 0 can be obtained. AD has been found to be associated with the marker allele ε4 in Africans, Asians, and Caucasians. This suggests that the AD-ε4 allelic LD predates the divergence of peoples occurring 100 000 years ago. With the age of AD-ε4 allelic LD so estimated, the maximal distance was calculated to be 23.2 kb (mean 5.8 kb). Conclusions (1) A method is developed for LD mapping of susceptibility genes. (2) A mutation within the APOE gene itself, among others, is responsible for the susceptibility to AD, which is supported by recent evidence from studies using transgenic mice. Chin Med J 2002; 115(8): 1233-1240

Objective To establish an in vitro heterogeneous expression model of human CYP2E1 (hCYP2E1) cDNA and investigate the effect of the chemical carcinogenic N, N' -dinitrosopiperazine (DNP) on the expression of CYP2E1 . Methods Exogenous hCYP2E1 was introduced into the mouse derived NIH3T3 cells using the lipofectamine transfection technique. Integration of exogenous hCYP2E1 gene was identified by PCR and Southern blot. After treatment with various concentration of ethanol and DNP on the transfected NIH3T3 cell cultures, RT-PCR and Western blot was applied to detect the expression level of CYP2E1. Results Two cell clones with integration and stable expression of exogenous hCYP2E1 were obtained and designated as NIH3T3-2E1-A4 and NIH3T3-2E1-A8 respectively. The expression of both hCYP2E1 mRNA and protein products was promoted after either ethanol or DNP treatment. Conclusion The results suggested that the promoted expression of hCYP2E1 induced by DNP and/or ethanol is due to enhanced transcription. The mechanism of DNP carcinogenesis might be related to this in situ activated metabolism by CYP2E1. Chin Med J 2002; 115(8): 1229-1232

Objective To determine the expression of E-prostanoid1 (EP1) and F-prostanoid (FP) receptor mRNAs in iris-ciliary bodies of the human eye using in situ hybridization. Methods EP1 and FP receptor mRNAs were detected by riboprobes labeled with digoxigenin on paraffin sections of the iris-ciliary body tissue of the human eye using in situ hybridization. Results EP1 and FP receptor mRNAs were highly expressed in blood vessels, muscles and the endothelia of the iris. EP1 receptor hybridization signals were present in all muscle fibers of the ciliary body. Hybridization signal corresponding to FP receptor mRNA transcript was predominantly expressed in the circular muscle and in the collagenous connective tissues of the ciliary body. FP receptor mRNA was not detected in radial and longitudinal muscles. Conclusions EP1 and FP receptor mRNAs in human ocular tissues appear to be widely localized in the functional sites of the respective receptor agonists. Selective localization of EP1 and FP receptor mRNAs in the circular muscles and collagenous connective tissues of the ciliary body suggests that EP1 and FP receptors play an important role in enhancing uveoscleral outflow of aqueous humor. Chin Med J 2002; 115(8): 1226-1228

Objective To investigate telomerase gene expression in precancerous mammary lesion, such as atypical ductal hyperplasia and breast cancer and to study the relationship between expression and malignant transformation. Methods Expression of human telomerase genes (hTR) and human reverse transcriptase gene (hTRT)in 76 cases of mammary tissue was evaluated using in situ hybridization and included 50 cases of mammary hyperplasia, 6 of which were benign hyperplasia, 9 were mild atypical hyperplasia, 12 were moderate atypical hyperplasia, 23 were severe atypical hyperplasia and 26 were mammary cancer. Results The expressions of hTR and hTRT mRNA were much weaker or negative in benign hyperplasia(16.6%, 0), weak to mild-moderate in atypical hyperplasia (22.2%, 11.1 %, 33.3%, 25.0%), strong in severe atypical hyperplasia (60.9%, 52.1 %). and significantly strong in mammary cancer (88.5%,80.8%). The difference between mild-moderate atypical hyperplasia, invasive ductal carcinoma and severe atypical hyperplasia was significant (P <0.05) and the difference between severe atypical hyperplasia and intraductal carcinoma was not significant (P> 0.05). Conclusion Telomerase genes (hTR and hTRT) expressions are related to the transformation of atypical hyperplasia. Activated telomerase may play a role in mammary cancer development. Chin Med J 2002; 115(8): 1221-1225

Objective To observe the unique DNA profile and the relationship between DNA profile and phenotype of Trichophyton rubrum, and establish an effective molecular method to identify T. rubrum. Methods Three primers, including (GACA)4, (GTG)5 and M13 core sequence (5'-GAGGGTG-GCGGTTCT-3'), were used to distinguish variations among 20 clinical isolates of T. rubrum and Trichophyton mentagrophytes . Results Different PCR-fingerprinting was seen between T. rubrum and T. mentagrophytes using three different primers. 2 stains of T. rubrum were identified among 6 supposed T. mentagrophytes. Conclusions T. rubrum can be distinguished using PCR, and (GACA)4 is the most suitable primer. Chin Med J 2002; 115(8): 1218-1220

Objective To develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies. Methods Using recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mlL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy,respectively . Results A new kind of gene-viral vector system of adenovirus, in which the E1 b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene therapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mlL-12. Replication and proliferation of the virus carrying the mlL-12 gene in tumor cells were confirmed by electron microscopy. Conclusions Gene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment. Chin Med J 2002; 115(8): 1213-1217

Objective To verify whether the extracellular domain of kinase domain region (KDR) has anti-angiogenesis activity in vivo. Methods cDNA was cloned into adeno-associated virus (AAV) vector pSNAV and transfected to baby hamster kidney (BHK) cells. Recombinant AAV was obtained from the cell culture supernatant after adding helper virus. Recombinant AAV-infected human bladder cancer EJ cell line (EJ cells) were injected subcutaneously into Balb-c nude mice. Tumor specimens were removed from the mice, paraffin-embedded and sliced, then stained by immunohistochemistry. Microvessel density (MVD) was determined under a microscope. Results The tumor volume developed by EJ cells transfected with the extracellular domain of KDR was significantly smaller (1 . 70 ± 0 . 18 cm3) compared with that in the control (5 . 62 ± 0 . 67 cm3) (P <0 . 05),although tumor developed to be detectable on almost the same time (14.7 ± 2.4 days vs 14.1 ±3.2 days). Further, MVD in the experimental group was lower than that in the control (41.3±4.8 vs 6.2±2.1, P<0.05). Conclusion The extracellular domain of KDR could be expressed in nude mouse bladder cancer tissue and inhibit tumor angiogenesis. Chin Med J 2002; 115(8): 1209-1212

Objective To determine the efficacy of adenovirus-mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. Methods Cytosine Deaminase (CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell Iine-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 107 Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. Results Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 1011 pfu/ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. Conclusions CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer. Chin Med J 2002; 115(8): 1205-1208

Objective To evaluate whether deletion of chromosome 14q is involved in the carcinogenesis of primary glioblastoma multiforme and to identify possibly common deletion regions. Methods Fourteen fluorescent dye-labeled polymorphic markers were used and polymerase chain reaction-based microsatellite analysis was employed to investigate loss of heterozygosity (LOH) on chromosome 14q in 20 primary glioblastoma multiforme (GBM). Results Ten of twenty (50%) GBM displayed LOH at one or more of the markers on chromosome 14q . Five tumors showed either LOH or non-informative on all markers tested. The most frequent LOH was observed at locus D1 4S65 (57.1%) on 14q32.1, and in the chromosomal region spanning from D14S63(47.1 %) to D14S74 (46.7%) on 14q23-31. None of the informative loci exhibited microsatellite instability. Conclusions Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM. Chromosomal regions at locus D14S65 on 14q32.1 and spanning from D14S63 to D14S74 on 14q23-31 may harbor multiple tumor suppressor genes associated with GBM. Chin Med J 2002; 115(8): 1201-1204

Objective To investigate the association of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene A/G polymorphism with susceptibility to diabetes mellitus in Han Chinese. Methods An A/G transition at position 49 of exon 1 was analyzed in 31 patients with type 1 diabetes, 31 patients with type 2 diabetes, and 36 controls were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. Results A highly significant increase in the frequency of the G allele was seen in patients with type 1 diabetes compared with controls (66.1 %vs. 34.7%, respectively; P <0.0005; OR=3.670). This reflected an increase in the GG genotype in patients (48.4%vs. 22.2%, respectively; P=0.025; OR= 3.281) and a significant decrease in the AA genotype (16.1 %vs. 52.8%, respectively; P=0.002). The allele frequencies of A and G in patients with type 2 diabetes were not significantly different from controls(A/G, 50.0/50.0%vs. 65.3/34.7%; P=not significant). The distribution of genotype, however,differed significantly. This difference reflected an increase in the AG genotype in patients (54.8%vs. 25.0%, respectively; P=0.012; OR=3.643) and a decrease in the AA genotype (22.6%vs. 52.8%,respectively; P=0.011). Conclusions CTLA-4 49 AA is protective from diabetes mellitus, whereas, CTLA-4 49 G allele (both as homozygotes and as heterozygotes) confers an increased risk of diabetes mellitus. Chin Med J 2002; 115(8): 1248-1250

Objective To provide insight into the psychosocial factors underlying the utilisation of health services by women with reproductive tract infection (RTI) symptoms. Methods A cross-sectional study, adopting Aday and Andersen's Social Behaviour Model, was conducted between 1998 and 1999 in Chinese Hebei province and Beijing. A total of 864 eligible married women (age 21 to 60 years) were face to face interviewed. Results The percentage of self-reported symptoms of RTIs in urban and rural women was 35.6 and 46.8, respectively; the proportion of women with RTIs who utilised health services was 27.5%and 26.7%, respectively. Compared to urban women, rural women had less knowledge on RTIs and more traditional beliefs, and were more satisfied with local health services. The results of logistic regression analysis showed that the common factor influencing health service utilisation in women with RTIs was current experience of RTIs. Knowledge about self-medication, perceived social stigma attached to RTIs, prior experience of RTIs, family income and perceived severity of RTIs were also predictors of utilisation of health services in rural women with RTIs. Satisfaction with health providers, information received from health providers, prior experience of RTIs, occupation and medical care coverage were predictors of utilisation of health services in urban women with RTIs. Conclusion The prevalence of RTIs is high, but the rate of seeking health services is low. There is a great need for emphasizing culturally acceptable reproductive health education in different places to improve women's ability for self-care. Regular medical check-ups for women are also important. It is necessary to improve the quality of health service, complete the reform of health insurance and alleviate women's social stigma related to RTIs, giving women social and moral support. Chin Med J 2002; 115(8): 1253-1256

Objective To review the current regional distribution of echinococcosis in China for the purpose of providing the scientific data for the control of echinococcosis in the future. Data sources All data in this paper were originated from related references in Chinese or English language Chinese journals except one from Lancet. Study selection Thirty-one original articles published from 1983 - 2001 were selected according to the stated purpose and 7 of them were written by the author. Data extraction The present paper addresses cystic echinococcosis (CE) and alveolar echinococcosis(AE), as well as their respective regional distribution. Results CE was distributed chiefly in Western China where Gansu, Xinjiang, Qinghai, Ningxia, Inner Mongolia, Tibet, west Sichuan and Qingzang plateau were hyper-endemic areas. Regional distribution of AE was not so extensive as that of CE, but it was found mainly in northwest China as well. Moreover, AE was extremely harmful to human body and "AE cancer" is popularly called. Conclusion Steps should be taken in the future heath policy to control its spread. Regional distribution of echinococcosis is so extensive in China that CE and AE should be increasingly controlled today. Chin Med J 2002; 115(8): 1244-1247