2000年 · 第113卷第11期

Objective To discuss the feasibility of gene therapy of human glioma by antiangiogenesis method. Methods Angiostatin K(1-3)cDNA with secretive signal was inserted into the polylinker sites of eukaryotic expression vector pcDNA3 to construct pcDNA-SAK(1-3). The vector was transfected into human SHG44 glioma cells by lipofectamine and the positive clone was screened by G418. The biological characteristics of glioma cells were examined by electronmicroscope and flow cytometry. The activity of angiostatin K(1-3)protein expressed by SHG44 cells was examined by the bovine micrangium endotheliocyte inhibition assay and immunofluorescence assay. When SHG44 cells were implanted into the strata subcutaneum of nude mice, tumor necrosis and micrangium were calculated immunohistochemically and electronmicroscopically for determining their characteristics and validity in gene therapy of human glioma by antiangiogenesis method. Results The eukaryotic expression vector pcDNA-SAK(1-3)was successfully constructed and transfected into glioma cells. The cells expressed angiostatin K(1-3)protein, and their tumorigenesis and angiogenesis in nude mice were greatly reduced. Conclusion Angiostatin K(1-3)gene is feasible to treat human glioma. This experiment lays a foundation for gene therapy of the other solid tumors by antiangiogenesis method. Chin Med J 2000; 113(ll): 996-1001

Objective To investigate the effects of ultraviolet blood irradiation and oxygenation(UBIO)on free radicals and antioxidase after spinal cord injury in rabbits. Methods Totally, 186 rabbits were used and divided randomly into four experimental groups: control(n = 6), blood transfusion(n = 24), injured(n = 96)and treatment(n = 60)groups. The relative intensity of free radical(FR)signals, malondialdehyde(MDA)content, as well as the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX)were compared among the four groups at 6, 24, 48, and 72 hours and 6 days after injury. Results The relative intensity of FR signals in spinal cord tissue in the injured group increased at 48 hours and showed a striking difference compared with the control group; in the treatment group, it decreased and showed a striking difference compared with the injured group. MDA content in blood in the injured group increased and showed a striking difference at 6, 24 and 48 hours and showed a significant difference at 72 hours and 6 days after injury compared with the control group. In the treatment group, MDA content in blood decreased and showed a significant difference at 48 hours compared with the injured group. MDA content in spinal cord tissue increased in the injured group and showed a striking difference compared with the control group; in the treatment group, it decreased and showed a striking difference compared with the injured group at the corresponding times. The activity of SOD in blood and spinal cord tissue decreased in the injured group and showed a striking difference compared with the control group; in the treatment group, it increased and showed a striking difference compared with the injured group at the corresponding times. The changes in activity of GSH-PX in blood and spinal cord tissue were similar to that in SOD. No significant difference was observed between the blood transfusion and control groups. Conclusion UBIO can ease free radical damages and elevate the activity of antioxidases after spinal cord injury in rabbits. Chin Med J 2000; 113(11): 991-995

Objective To investigate the level of telomerase activation(TA)and telomerase catalytic subunit(hEST2)gene mRNA in patients with non-small cell lung cancer, and determine whether they are associated with tumor cell apoptosis, stage, and clinical outcome. Methods Primary tumor specimens from 58 patients untreated with chemotherapy and 10 cases of histologically benign and adjacent lung tissue were analyzed. TA and hEST2 were measured by means of a modified telomerase repeat amplification protocol(TRAP)assay and in situ hybridization(ISH), respectively. Terminal deoxynucleotidyl transferase(TdT)-mediated biotin dUTP nick end-labeling(TUNEL)was used to evaluate apoptotic cells. Reverse transcription polymerase chain reaction(RT-PCR)was used to detect bcl-2 mRNA expression. Results TA and hEST2 were detected in 45(77.6%)and 43(74.1 %)of 58 tumor specimens, respectively, and not detected in specimens of adjacent and benign lung tissue. One case expressed hEST2 as a weak positive. Statistically significant positive association was found between the level of TA and hEST2(r = 0.85, P = 0.001). TA and hEST2 were associated with tumor stage, but not associated with tumor grade, gender and patient age. Positive rate of bcl-2 mRNA was 38(65.5%)of 58 tumor specimens. The mean apoptotic index in the bcl-2 positive group(9.5±1.3)was lower than that in the bcl-2 negative one(19.8±2.1, P<0.05), suggesting that apoptotic index may be inversely associated with bcl-2 expression(r=-0.48, P= 0.041). Bcl-2 expression in the TA and hEST2 positive group(92.1% and 89.4%)was higher than that in the negative one(50.0% and 45.0%, P = 0.043 and P = 0.032, respectively). The apoptotic index was lower in the TA or hEST2 positive group(8.2 ±1.4, 10.7±1.1)than in the negative one(20.5± 1.6, 24.2 ± 2.1, P < 0.05). A statistically significant inverse association was found between TA or hEST2and apoptotic index(r =-0.45, P = 0.02 and r =-0.51, P =0.001, respectively). Positive correlation was also detected between TA or hEST2 and bcl-2 expression(r = 0.86, P = 0.01 and r = 0.73, P = 0.024, respectively). The level of hEST2 mRNA and apoptotic index were associated with clinical outcome in a multivariate cox regression analysis. Conclusions High TA and hEST2 were frequently detected in primary non-small cell lung cancer untreated with chemotherapy, having high bcl-2 expression and a low tumor cell apoptotic rate. This suggests that both TA and hEST2 are correlated with the deregulation of apoptosis. hEST2 and apoptotic index have prognostic significance in patients with non-small cell lung cancer. Chin Med J 2000; 113(11): 985-990

Objective To approve a theoretical basis for the molecular pathogenesis of human cerebral malaria and treatment with prevention. Methods The blood samples were collected from 24 patients with cerebral malaria, 143 with falciparum malaria, 34 with vivax malaria and 20 healthy controls from the endemic areas of Yunnan Province, China. Using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)technique, we determined the molecular mass(Mr)of these Plasmodium falciparum(P. falciparum)erythrocyte membrane protein 1(PfEMPl)molecules. Results Our findings indicate that higher molecular mass(260 kDa-320 kDa)forms of PfEMPl were expressed on parasitized erythrocyte(PE)from human cerebral malaria patients. Compared with PfEMPl expressed on PE from human cerebral malaria patients, the expression of PfEMPl and Plasmodium vivax(P. vivax)erythrocyte membrane protein 1(PvEMP1)on PE from falciparum malaria patients and vivax malaria patients did not have multiple bands of PfEMPl of ≥ 260 kDa, but had a PfEMPl with molecular mass of 240 kDa and a PvEMPI with molecular mass of 180 kDa band separately. Healthy controls expressed an EMP of molecular mass of 140 kDa. Conclusion Results confirm the antigenic variation of higher molecular mass of PfEMPl whose molecular mass is equal to or exceeds 260 kDa-320 kDa on PE of patients with cerebral malaria. Our results show that the binding of large antigenic variability PfEMPl molecular mass of 260 kDa-320 kDa on PE from human cerebral malaria patients with diverse receptor molecules on the endothelial cell(EC)of the cerebral microvessels may be involved in the molecular pathogenesis of cerebral malaria. Chin Med J 2000; 113(11): 981-984

Objective To investigate the role of bax in a vincristine(VCR)-induced multidrug-resistant(MDR)human gastric cancer cell line, SGC7901/VCR, in which the Bax protein expression level was significantly lower compared with that in parent cells. Methods A bax eukaryotic expression vector was constructed and transfected into SGC7901/VCR cells by lipofectamine, and resistant clones were selected by G418. Western blotting detected Bax expression in transfectants. Tetrazolium blue(MTT)assay evaluated the differences in drug sensitivity and cell cycle changes of transfectants were analyzed using flowcytometry(FCM). Results The bax eukaryotic expression vector was constructed and transfected into SGC7901/VCR cells. Through G418 selection, resistant clones were obtained. Western blotting demonstrated that the expression of Bax protein was markedly increased in bax transduced cells. These cells were more sensitive to adriamycin(ADR)and VCR than mock vector transducted cells. Moreover, bax transfection enhanced ADR-induced apoptosis and VCR-induced G2/M phase arrest of SGC7901/VCR cells. Conclusion Bax was involved in the MDR of SGC7901/VCR cells. Chin Med J 2000; 113(11): 977-980

Objective To determine the mechanism of all-trans retinoic acid(ATRA)on growth inhibition in human gastric cancer cells. Methods Gastric cancer cell lines: MGC80-3, BGC-823, SGC-7901 and MKN-45. CAT assay, Northern blot, Western blot, gene transfection and MTT assay. Results ATRA can inhibit the activator protein-1(AP-1)activity in ATRA-sensitive cell lines, but not in ATRA-resistant cell line, and the anti-AP-1 activity of ATRA is mediated by its receptor, retinoic acid receptor a(RARα). ATRA can also inhibit the expression of cJun and cFos. One of the mechanisms for ATRA to inhibit the growth of gastric cancer cells may be through its inhibitory effect on the AP-1 activity and its influence on up-regulation of RARα expression. The inhibition of cJun and cFos expressions by ATRA may also contribute to the anti-AP-1 activity. Conclusions ATRA inhibits the growth of gastric cancer cells through the regulation of AP-1 activity. This action is mediated by RARα. Chin Med J 2000; 113(ll): 972-976

Objective To evaluate the feasibility and short term results of transcatheter closure of secundum atrial septal defects(TCSASDs)using Amplatzer occluder device. A new self-centering device, the Amplatzer atrial septal occluder, has recently been evaluated in an animal model with excellent results. This paper reports our clinical research with this device. Methods 30 patients underwent an attempt at TCSASDs at an average age of 35.6 ± 15.9 y(range, 5-62 y)and average weight of 63.6±14.5 kg(range, 17-78 kg)using the Amplatzer atrial septal occluder. The average ASD diameter measured by transesophageal echocardiography(TEE)was 23.6± 4.0 mm(range, 16-31 mm), and the average atrial septal defects(ASD)balloon stretched diameter was 25.2 ± 5.8 mm(range, 9-34 mm). All patients had right atrial and ventricular volume overload with a Qp/Qs of 3.4±1.2(1.4-5.2). A F8-12 catheter was used for delivery of the device in all patients. Results The device was placed correctly in all patients. There was immediate and complete closure in 30 patients. The average device waist size used to close the defect was 25.6 ± 5.9 mm(range, 9-34 mm). There was no episode of device embolization or any other complication. Follow-up was performed using transthoracic echocardiography(TTE)1 day, 1 month, 3 months, 6 months and 1 year after the procedure. The first day after operation; there was complete closure of the ASD in all patients; there have been no episodes of endocarditis, thromboembolism, or wire fracture. Conclusion TCSASDs using the Amplatzer occluder device was an efficient nonsurgical method which had a very high technical success rate and satisfactoy short term and follow-up results. Chin Med J 2000; 113(ll): 967-971

Objective To explore the relationship between genetic variation in coagulation factor V and the occurrence of coronary arterial disease(CAD). Methods Unrelated 86 patients with CAD and 102 healthy controls were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)to detect variations in the entire twenty-five exons of the factor V gene. Results Polymorphisms in exon 4[642 G → T(Ser156)], exon 10 [1628 G→ A(Arg485Lys)], exon 13 [4070 A→ G(His1299Arg)] and exon 16 [5380 G→A(Val1736Met)] were documented. The study also identified a novel polymorphism in exon 2(327 A→G)which did not result in amino acid residue substitution. The Leiden mutation(Arg506Gln)was not detected in any of our 188 subjects. Among the 5 polymorphisms, the allele frequency of 1628 G → A was significantly different between CAD patients and controls(0.69 vs 0.81, x2 = 6.908, P <0.01). This is the first report of this finding in a Chinese population. Conclusion 1628 G→ A polymorphism is associated with CAD and it may be a risk factor for CAD morbidity in the Chinese population. Chin Med J 2000; 113(11): 963-966

Objective To determine whether cholinergic neurons in the basal nucleus magnocellularis(NBM)and the medial septum are affected in transgenic mice overexpressing human amyloid precursor protein 770 (APP770). Methods Eight age groups, from 3 months old to 10 months old, of either heterozygous transgenic or non-transgenic mice were used for choline acetyltransferase(ChAT)staining using immunohistochemistry. The number of ChAT-positive neurons was counted on the MCID Image Analysis System. Neurons in the cerebral cortex and area CA1 of hippocampus were also stained with cresyl violet and counted using optical dissector technique. Results There is no change in the number of forebrain cholinergic neurons in the transgenic mice up to 9 months of age. A loss of these cholinergic neurons starts in 9 months old transgenic mice, with a further decrease in the number of NBM and medial septum neurons in 10-month-old transgenic mice. On the other hand, the number of neurons in the cerebral cortex and hippocampal area CA1 remained unchanged. Conclusion These results demonstrate a selective loss of basal forebrain cholinergic neurons in APP770 transgenic mice. Chin Med J 2000; 113(11): 1040-1042

Objective To study the clinicopathologic features and immunoprofile of angiomyofibroblastoma(AMF)with emphasis on differential diagnosis. Methods Seven vulvar, two vaginal and one perineal angiomyofibroblastomas were evaluated by light microscopy and immunohistochemistry. The immunohistochemical studies were performed on paraffin-embedded sections using the standard ABC method with a panel of antibodies. Results Angiomyofibroblastoma predominantly occurred in middle-aged women in the genital region, especially in the superficial area of the vulva. Clinically, most of the tumors presented as slowly growing painless masses and were often diagnosed as Bartholin's gland cysts. Histologically, the tumors were all well-circumscribed and characterized by alternating hypocellular and hypercellular areas with abundant thin-walled blood vessels. The tumor cells were bland and spindle-shaped or epithelioid and tended to concentrate around the vessels or cluster in small nests. Immunohistochemically, the tumor cells expressed vimentin and estrogen receptor protein in all 10 cases and desmin in 9 cases. Three cases showed weak or focal immunoreactivity to alpha-smooth muscle actin and muscle specific actin. All ten patients were treated by local excision. Follow-up showed a benign course with no signs of recurrence. Conclusions Angiomyofibroblastoma is a distinctive neoplasm that has a propensity to occur in the female genital tract. Recognition of this entity is important to avoid misdiagnosis with other vulvar angiomyxoid neoplasms. Chin Med J 2000; 113(11): 1036-1039

Objective To evaluate the role of p27Kip1 in tumorigenesis and the development of trophoblastic cell disease. Methods Using immunohistochemistry, the expression of p27-protein was investgated in 10 normal chorionic villi in the first trimester of pregnancy, 15 complete hydatidiform moles(HM), 7 invasive moles(IM)and 7 choriocarcinomas(CC). Results In all cases, immunohistochemical staining localized p27-protein in the plasma. Decreased expression of p27Kip1 was observed in malignant trophoblastic neoplasms with a positive rate of 21.43％, which is significantly less than that in normal chorionic villi(80%)and in complete HM(73.33%)(P < 0.05). The positive rate of p27Kip1 in those complete HM with large uterine size for gestational age was lower than that in those with normal or small uterus(42.86% vs 100%, P < 0.05). Conclusion p27Kip1 may be involved in the tumorigenesis of gestational trophoblastic neoplasm as a negative regulator of the cell cycle. The expression level of p27Kip1 in trophoblastic cells may be a prognostic factor for complete HM. Chin Med J 2000; 113(11): 1046-1048

Objective To describe the etiologies, clinical features and diagnosis of cerebral venous sinus thrombosis. Methods We reviewed the records of 23 patients admitted with a documented diagnosis of cerebral venous sinus thrombosis from 1991 through 1999 in the Beijing Tiantan Hospital. Results Infection was the major condition associated with cerebral venous sinus thrombosis. Pseudotumor cerebri syndrome was the most common manifestation, while hemiplegia, seizure and unconsciousness may occur alone or in association. The diagnostic sensitivity of computed tomography(CT), magnetic resonance imaging(MRI)and digital subtract angiography(DSA)were 59%, 86% and 100%, respectively. The sensitivity of MRI with magnetic resonance angiography(MRA)reached 96%. Conclusion As the clinical findings were found to be nonspecific, CT combined with DSA, or MRI with MRA turns out to be valuable for the early diagnosis of cerebral venous sinus thrombosis. Chin Med J 2000; 113(11): 1043-1045