2004年 · 第117卷第03期

Background In humans telomerase is expressed in most cancers and immortal cell lines, and astivation of telomerase may play important roles in tumorigenesis and immortalization. This study was to investigate the roles of telomerase activity (TA) and human telomerase RNA (hTR) in sebaceous carcinoma of the eyelid. Methods The telomerase repeated amplification protocal (TRAP) was used to demonstrate telomerase activity in 12 cases of sebaceous carcinoma of the eyelid. In situ hybridization (ISH) was used to demonstrate the expression of hTR in 55 cases of paraffin-embedded sebaceous carcinoma of the eyelid, and the results were compared with the proliferative index determined by Mib-1 immuno-labeling, histological patterns and recurrence of the tumor. Results Different telomerase activity was shown in the 12 cases of sebaceous carcinoma of the eyelid. The positive expression of hTR was 85. 5%(47/55) in tumor cells, but not in the adjacent tissues. The positive expression of hTR was correlated with the proliferative activity (as assessed by Mib-1 immunolabelling, r=0. 942, P <0. 001) and the differentiation of sebaceous carcinoma of the eyelid (χ2=17. 621, P <0.001), but not significantly related to tumor recurrence. The level of hTR expression increased with the decrease of differentiation of sebaceous carcinoma of the eyelid. Conclusions The results suggest that the up-regulation of telomerase expression plays some roles in tarsal gland carcinogenesis, and the expression of hTR is a useful marker for malignant degree of sebaceous carcinoma of the eyelid. Chin Med J 2004; 117(3): 445-448

Background We investigated the expression and role of TN4 in the oncogenesis of human hepatocellular carcinoma (HCC) from Qidong which is a HCC risk area. Methods The expression of TN4 in HCC was observed using immunohistochemical staining (IHC). TN4 levels were manipulated in human liver cancer cell SMMC7721, using pcDNA3. 1 eukaryotic expression constructs designed to express the complete TN4 cDNA. The biological changes of the cells were observed before and after transfection of TN4 and the change of gene expression was analysed by atlas cDNA expression array. Results Among 100 pairs of samples of HCC, TN4 down-regulation expression and up-regulation expression positive rate were 81%(81/100), 19%(19/100), respectively (P <0. 01). TN4 protein was mainly localized in cytoplasm and membrane. The positive rate of TN4 were 10%(3/30), 100%(70/70) in lymph node metastasis and no lymph node metastasis, respectively (P <0. 01). The growth rates of the derivative SMMC7721-TN4 cell lines were decreased in comparasion with that of normal SMMC7721 cells and pcDNA- SMMC7721. Some gene expression was changed before and after transfection of TN4. At 30 days of post-implantation of SMMC7721-TN4, SMMC7721-pcDNA3,SMMC7721 group produced tumors of (301. 9 ±143. 4) mm3,(2418. 7 ±362. 8) mm3,(2317. 4 ±587. 8) mm3, respectively, (P<0. 01). Tumor inhibiting rate was 82. 4%in TN4 transfection group. Sections of tumors were observed for their degree of tissue necrosis and there was higher degree of necrosis in tumors of the TN4-SMMC7721 cell group than those of the SMMC7721, SMMC7721 -pcDNA groups. Conclusions TN4 may play an important role in the oncogenesis of human HCC, especially in Qidong, the HCC risk area and TN4 could be a candidate tumor suppressor gene for HCC. Chin Med J 2004; 117(3): 440-444

Background RevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficiency (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On. Methods Herpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR). Results MCF/TRE/tk/Tet-On cell survival rate was decreased from 100%to less than 20%when ganciclovir (GCV) concentration was increased from 0 to 1000 μg/ml at 1 μg/ml of Dox after 72 hours of GCV administration. At 1 μg/ml of GCV concentration, the cell numbers decreased from 7 × 104cells/ml to 2 × 104 cells/ml when Dox concentration was increased from 0 to 1500 μg/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10%-25% of transduced MCF-7cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control. Conclusion The human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo. Chin Med J 2004; 117(3): 434-439

Background We investigated the co-expression of calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV, a combination of the three is referred to as CaBPs) with y-aminobutyric acid(GABA) or glycine in neurons of the rat medullary dorsal horn (MDH). Methods Immunofluorescence histochemical double-staining for CaBPs and GABA or glycine was performed on the sections from rat MDH. Results CB-, CR-, PV-, GABA- and glycine-like immunoreactive (LI) neurons were differentially observed in all layers of the MDH, but particularly in lamina II . Neurons that exhibited immunoreactivity for both CaBPs and GABA or glycine were also observed mainly in lamina II. A few of them were found in laminae I and III. The percentages of neurons which co-expressed CB/GABA or CB/glycine out of the total numbers of CB- and GABA-LI neurons or CB- and glycine-LI neurons were 5. 3% and 12. 1% or 4. 1% and 10. 0%, respectively. The ratios of CR/GABA or CR/glycine co¬existing neurons out of the total numbers of CR- and GABA-LI neurons or CR- and glycine-LI neurons were 5. 8% and 7. 6% or 4. 4% and 7. 1%, respectively. The rates of PV/GABA or PV/glycine co-localized neurons out of the total numbers of PV- and GABA-LI neurons or PV- and glycine-LI neurons were 11. 1% and 5. 1% or 9. 9% and 5. 1 %, respectively. Conclusion The results indicate that some neurons in the MDH contain both CaBPs and GABA or glycine. Chin Med J 2004; 117(3): 430-433

Background Adipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons. Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE). Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies. Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation. Chin Med J 2004; 117(3): 425-429

Background To investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse. Methods The recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 105 EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with Ads Fas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT. Results The adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2. 5 × 1011 pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P>0. 05), but indices in Group A were significantly lower than those in the latter three groups (P <0. 01). Counts of leukocytes and platelets on+30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100%in Group B (6/6)and D (16/16), 12.5%(2/16) in Group C and 6. 25%(1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P<0. 01). Conclusions The transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT. Chin Med J 2004; 117(3): 419-424

Background We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia(GVL) effects and to investigate the distribution of T-cell receptor (TCR) Vβ gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction(PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR Vβ genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Results The 24 TCR Vβgene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Another 4 donors expressed part of the 24 TCR Vβsubfamily and 1 donor had oligoclonality. The expressions of the 24 TCR Vβ subfamilies were skewed and restricted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR Vβsubfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6 -30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in Vβ 2 -3, 16 -17, 18 -19, 21 and Vβ 23 in patients with GVHD and in Vβ 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had Vβ 15 and 16 T cell expansion after transplantation. One patient displayed Vβ 18 T cell expansion after donor lymphocyte infusion (DLI). Conclusions Normal individuals express the entire 24 TCR Vβ gene repertoire and have polyclonal distribution. However, the TCR Vβ gene repertoire is only partially expressed in some donors. The TCR Vβ gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR Vβgene subfamilies increases over time post¬transplantation. GVHD and GVL effects may induce the proliferation of T cell clones. Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen. Chin Med J 2004; 117(3): 413-418

Background We investigated the role of 1, 25-dihydroxyvitamin D3 (1, 25-(OH)2D3) in preventing allograft from acute rejection following orthotopic liver transplantation. Methods A rat orthotopic liver transplantation model was used in this study. SD-Wistar rats served as a high responder strain combination. Recipients were subjected to administration of 1, 25-(OH)2D3 at dosages ranging from 0. 25 μg • kg-1 • d-1 to 2. 5 μg • kg-1 • d-1. Survival after transplantation as well as pathological rejection grades and IFN-γ mRNA, IL-10 mRNA transcription intragraft on day 7,and day 30 post-transplantation were observed. Results After recipients were treated with 1, 25 (OH)2D3 at dosages of 0. 5 μg • kg-1 • d-1 or 1. 0μg·kg-1·d-1, survivals of recipients were prolonged. Ninety-five percent confidence intervals of survival were 46 -87 days and 69 -102 days (both P=0. 0005 vs control group), respectively. On day seven post-transplantation, relative levels of IFN-γ mRNA transcription were 0. 59 ±0. 12 and 0. 49 ±0. 16, which was higher than the control group (P=0. 005, P=0. 003, respectively). Relative levels of IL-10 mRNA transcription were 0. 83 ±0. 09 and 0. 76 ±0. 09, which was lower than the control group (P=0. 002, P=0. 003, respectively). At a dosage of 0. 5 μg • kg-1 • d-1, the median of pathological rejection grade on day seven and on day thirty post-transplantation were 1. 5 and 2. 0 in comparison with the CsA-treated group (P=0. 178, P=0. 171, respectively). At a dosage of 0. 5 μg• kg-1 • d-1, the median of pathological rejection grade on day seven and day thirty post-transplantation were 1. 5 and 1. 5 in comparison with CsA-treated group (P=0. 350, P=0. 693, respectively). Conclusion After each recipient was treated with 1, 25-(OH)2D3 at a dosage of (0. 5 -1.0)μg • kg-1 • d-1, transcription of cytokine intragraft was accommodated effectively and deviated to Th2type, resulting in alleviation of acute rejection. 1, 25-(OH)2D3 can prolong survival of recipient after orthotopic liver transplantation. Chin Med J 2004; 117(3): 408-412

Background The purposes of this study were to confirm the changes in myocardial collagen level after left ventricular assist device (LVAD) support in dilated cardiomyopathy (DCM), find the relation between these changes and prognosis, and test a practical method to assess the level of myocardial collagen. Methods Left ventricular samples were collected from DCM patients with different prognosis(transplanted group n=8, weaning group n=10) at the time when the LVADs were implanted and again during cardiac transplantation (n=8). The level of neutral salt soluble collagen (NSC) and acid soluble collagen (ASC) was measured by Sircol collagen assay, and that of total collagen and insoluble collagen (ISC) by quantification of hydroxyproline (Hyp). Serum samples were collected from a portion of these patients (transplanted group, n=6; weaning group n=7) at the time the LVADs were implanted, 1 month after implantation and on explantation. Circulating concentration of carboxy-terminal propeptide of type I procollagen (P I CP), amino-terminal propeptide of type I procollagen (P I NP), amino-terminal propeptide of type III procollagen (P III NP) and type I collagen telopeptide (I CTP) were measured by the equilibrium type radioimmunoassay. Results Before LVAD implantation the level of NSC and ISC in the weaning group was higher but ASC in the transplanted group was lower than in the controls (P <0. 05). After LVAD support, the level of total collagen was higher, but ASC was also lower in the transplanted group than in the controls (P <0. 05). In comparison of the pre- and post-LVAD subgroups of the transplanted and weaning groups, all collagen fraction levels before LVAD implantation were lower in the transplanted group than in the weaning group (P <0. 05); but this difference disappeared after LVAD support. Comparison of the pre- and post-LVAD subgroups of the transplanted group showed increased level of NSC and total collagen after LVAD support. The changes of serum peptide concentration showed that P III NP increased constantly in the transplanted group, but P I CP and P I NP increased in the weaning group after LVAD implantation. Conclusions The changes in myocardial collagen level as a sign of myocardial interstitial remodeling in DCM are not involved with total collagen but invalved with collagen fractions, and they are related to prognosis. The changes of myocardial collagen content and serum procollagen peptide after LVAD support can be regarded as an expression of the reverse of maladaptive myocardial interstitial remodeling. Chin Med J 2004; 117(3): 401-407

Background The activation of extracellular signal-regulated kinase1/2 (ERK1/2) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mKATP) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mKATP and ERK1/2 in the delayed protection mechanism of anoxic preconditioning (APC). Methods An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptopropionylglycine(MPG), and PD98059] were used to modulate mKATP and ERK1/2 activation. Cellular injury was evaluated by measuring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mKATP opener). Results Cell viability and SOD activity in the APC [(81. 9 ±11. 4)%, (13. 6 ± 3. 7) U/L] and diazoxide [(79. 2 ±12. 4)%, (16. 5 ±4. 6)U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42. 2 ±7.3)%,(8. 8 ±2. 8) U/L] group (all P <0. 01). LDH activity in the APC group [(101. 9 ±18. 9) U/L] and diazoxide group [(97. 5 ±17. 7) U/L] was significantly lower than in the A/R group [(250. 5 ±43. 6)U/L] (all P <0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK1/2 activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP(an mKATP blocker) and by MPG (a free radical scavenger). In addition, the ERK1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. Conclusion mKATP may mediate ERK1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes. Chin Med J 2004; 117(3): 395-400

Objective To evaluate the efficacy and safety of misoprostol in term labour induction. Data sources Data from published English and Chinese literatures about misoprostol in term labour induction were identified from Medline and CBMdisk (using the search terms "misoprostol" and "labour induction") before 2001; hand searches of reference lists of original studies and reviews(including meta-analyses) and contact with investigators in this field before 2001. Study selection Studies were included if they had data on misoprostol and labour induction. Altogether 623 articles were found and 124 were admitted, including 19 287 cases. Data extraction Data were collected on efficacy and incidence of side-effects of misoprostol and oxytocin. Data were checked for consistency within the published articles and converted into a standard format for incorporation into a central database. Data synthesis The average successful induction rate, rates of caesarean section; incidence of tachysystole, hypertonus of uterus and precipitous labour, and rates of meconium stained amniotic fluid between the misoprostol and oxytocin groups were significantly different (P <0. 05). There were no significant differences between the two groups concerning the average interval from the administration of misoprostol and oxytocin to the onset of labour, duration of the total stage of labour,incidence rate of foetal distress, neonatal asphyxia (1 -minute Apgar score ≤ 7), postpartum haemorrhage or amount of blood loss in postpartum. Conclusions Misoprostol is a superior agent over oxytocin on the induction of term labour, but its application might increase the risk of precipitatous labour, abnormal uterine contractions or meconium stained amniotic fluid. Therefore, the doasges and regimens of the agent need further investigation. Chin Med J 2004; 117(3): 449-452