2001年 · 第114卷第07期

Objective To investigate whether artificial heterozygous chimeras of platelets can be established by intrauterine transplantation of fetal liver stem cells and evaluate its potential use for the treatment of Glanzmann thrombasthenia. Methods Platelet glycoprotein (GP) II b Bak a/b (or GP II b I le843Ser) was used as a genetic marker. A homozygous 16-week-old Bak a/a fetus (as donor) and a homozygous 16.5-week-old Bak b/b fetus (as recipient) were screened from 42 pregnant women hospitalized for abortion. PGR with allele specific primers and FOK I digestion based on PCR products were used. Aborted donor fetal liver cell suspensions were prepared and intrauterine transplantation was carried out by infusion of 4 ml fetal liver cells (22 × 105) into the recipient umbilical vein under ultrasonic visualization. Results At gestation termination (abortion), 21 days after transplantation, chimera GP Ⅱb Bak a/b of the recipient were detected by FOK 1 digestion based on PCR from DNA and RT-PCR from platelet RNA. Conclusion Intrauterine transplantation of fetal liver cell may provide an effective way for curing GT or other inherited diseases. Chin Med J 2001; 114(7):676-679

Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model. Methods Human CD40 gene extracellular region was inserted into plasmid plG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated. Results Mammalian expression vectors pIG/40Ig and p3.1/40lg were constructed as described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease. Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease. Chin Med J 2001; 114(7):685-689

Objective To develop a retroviral-mediated high efficient expression system of human coagulation factorⅧ. Methods The LNC-FⅧ BD retroviral vector was generated by cloning a human B-domain-deleted (760aa ~1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧ BD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells• 24 hrs-1, and the FⅧ antigen was 500 ng/106 cells•24 hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12 U/106 cells•24 hrs-1 and 62.4 ng/106 cells•24 hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells. Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A. Chin Med J 2001; 114(7):690-693

Objective To evaluate the effects of cyclosporin A (CsA) on the apoptosis of bone marrow cells of aplastic anemia (AA) patients. Methods The apoptosis of and Fas antigen expression on bone marrow mononuclear cells (MNC) in 25 AA patients and 10 normal controls were assayed by TUNEL and FACS, respectively. Results The percentage of CD34+ cells was decreased significantly in AA patients compared with normal controls (P < 0.05), as was the percentage of CD34+ Fas+ cells (P < 0.05). The percentage of apoptotic cells in AA patients was higher than that of the controls (P < 0.01). After the patients were treated with CsA for 2 months, the percentage of apoptotic cells in AA patients decreased. Conclusion Fas is involved in the apoptosis of CD34+ cells of AA patients. CsA could reduce the percentage of appoptosis of bone marrow cells in AA patients. Chin J Hematol 2001; 22: 189-191

Objectives To evaluate the value of detecting p53 gene point mutations in sputum samples and its validity and reliability as a surveillance index in the early diagnosis of lung cancer in suspicious patients. Methods Sputum samples were collected from 54 cases identified as lung cancer and 114 cases identified as pulmonary benign disease. The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was performed for the detection of point mutations at exons 5-8 of the p53 gene, and sputum smears were also used for each sample. Results The detected mutation rate of 55.56% (30/54) in the lung cancer group was significantly higher than that of 1.75% (2/114) in the control group (P < 0.001). In the lung cancer group, the sensitivity, specificity, and positive likelihood ratio (PLR) of p53 mutations as a diagnostic marker for lung cancer were 55.56%, 98.25% and 31.75%, respectively. Also, the detection rates were 35.19% (19/54) by smears and 55.56% (30/54) by PCR-SSCP-silver stain, respectively. The differences both in rate and consistency were statistically significant (P < 0.01 and P < 0.05, respectively). Moreover, statistical analysis showed no significant relations between p53 mutations and clinical parameters such as gender, smoking habits, histotypes and stages, but the detection rate of p53 mutations in older patients (≥60 years old) was significantly higher than that in younger patients (P = 0.02). One case with p53 mutations at exon 5 in the control group was confirmed to be squamous cell carcinoma after 4 years of follow-up. Conclusion Detection of p53 gene alterations in sputum samples by PCR-SSCP-silver stain can be used as a follow-up surveillance index for the early diagnosis of lung cancer in suspicious patients. Chin Med J 2001; 114(7):694-697

Objective To analyse the relationship between age and treatment with captopril after acute myocardial infarction (AMI). Methods In a randomized trial, 822 patients with a first AMI received conventional medical treatment, including intravenous thrombolytic therapy and oral aspirin or metoprolol, and then were randomly allocated to captopril [dosage from the first 6.25 mg to 25 mg/t.i.d, 209 younger patients (≤64 years), 269 elderly patients (65-75 years)] or conventional treatment only (131 younger patients, 213 elderly). Survival in the four groups was calculated with the Kaplan-Meier method. Multivariate analysis was performed to understand the degree that multi-variables (including age) affect survival in patients taking captopril in the hospital or during long term follow-up. Results The survival of patients who took captopril correlated significantly with age (P < 0.001). The survival of the elderly patients on captopril treatment did increase (P < 0.0001), but not of the younger ones (P > 0.05) during hospitalization. During follow-up, the survival of patients who took captopril correlated insignificantly with age (P > 0.05), but both the elderly and the younger patients have good survival rates (all P<0.01) and lower cardiac events (all P < 0.01) when they took captopril. Conclusions Captopril exerts a weak effect on the younger patients but a beneficial effect on the elderly patients during hospitalization after AMI. However, there is no difference between the younger and the elderly in the prognosis, both having good survival and lower cardiac events when they take captopril long term during follow-up. Chin Med J 2001; 114(7):698-702

Objective To explore the therapeutic effectiveness of the combination of cyclosporin A (CsA) and androgen in the treatment of chronic aplastic anemia (CAA). Methods Androgen alone or combined with CsA for the treatment of CAA was compared by a randomized controlled clinical trial. Results The efficacy of androgen combined with CsA (87.9%) was higher than that of androgen alone (57.1%). Therapeutic effectiveness of the combination treatment between the patients with positive and negative peripheral blood mononuclear cells (PBMNCs) inhibiting normal colony formation unit-granulocyte and macrophage (CFU-GM) test showed a significant difference (P < 0.005). Conclusions Androgen combined with CsA has a much better efficacy than that of androgen alone in the treatment of CAA. The patients' PBMNCs inhibiting normal CFU-GM growth test can be used as an index of the treatment outcome. The side effects of the combination therapy are low and tolerable. Chin J Hematol 2001; 22: 186-188

Objective To investigate the correlation among vacA genotypes, cagA gene, VacA, serum CagA antibodies of Helicobacter pylori (H. pylori) and gastroduodenal diseases. Methods vacA genotypes and cagA gene of 62 H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were tested by polymerase chain reaction, and Hela cell assay for VacA activity in vitro. Serum CagA antibodies were measured by EIA method in the same patients. Results All 62 H. pylori strains possessed the vacA gene and vacA genotypes of all strains were type s1a/m2. Total positive rate of cagA gene was 56.45%; the positive rates of cagA gene of H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were 55.56%, 54.17%and 63.64%, respectively (P > 0.05). The total positive rate of VacA was 37.10%; the positive rates of VacA produced by H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were 33.33%, 29.17% and 63.64%, respectively (P > 0.05). The positive rates of CagA antibodies in patients with chronic gastritis, peptic ulcer and gastric cancer were 70.37%, 79.17% and 40.00%, respectively (P > 0.05). The total positive rate of CagA antibodies was 68.85%. Conclusion There was no correlation among cagA gene and vacA genotypes of H. pylori, VacA, serum CagA antibodies and various gastroduodenal diseases. Chin Med J 2001; 114(7):703-706

Objective To explore more effective regimen for reducing early mortality of severe aplastic anemia (SAA) and improving therapeutic effectiveness. Methods Antilymphocyte globulin/antithymocyte globulin (ALG/ATG) and cyclosporine A (CsA) (sequential intensified immunosuppressive therapy, SIIST), with or without hematopoietic growth factors (HGFs) were administered to 73 SAA patients in a prospective randomized clinical trial to test the effectiveness of the addition of HGFs for the patients. Results The response rate of the SIIST with HGFs group was significantly higher than that of the SIIST alone group (89.2% vs 63.9%), with lower rates of early infection (24.3% vs 55.3%) and mortality (4.0% vs 16.7%), shorter duration of cytopenia and blood transfusion dependence and faster recovery of bone marrow hematopoiesis. The addition of HGFs to SIIST was tolerated well in all patients. There was no difference in the treatment outcome of the two groups with GM-CSF plus Epo or G-CSF plus Epo. Conclusion The use of HGFs in combination with SIIST could reduce early infection and mortality rates, and therefore, improve the response rates in SAA patients. Chin J Hematol 2001; 22: 182-185

Objective To establish an effective exercise prescription, the exercise capacity of obese subjects was investigated. Methods In 71 male obese (body mass index: BMI ≥ 26.4) Japanese, aged 26-77 years, the body composition and physical fitness such as aerobic exercise level, muscle strength and flexibility were determined and compared with those of age- and sex-matched 71 borderline (24.2 ≤ BMI < 26.4) and 71 normal Japanese men (19.8 ≤ BMI < 24.2). The relationship between body composition and other parameters was evaluated in obese subjects. Results In obese subjects, body weight, BMI, body fat percentage, waist hip ratio, and skinfold thickness were higher than those of borderline and normal subjects. Lower aerobic exercise level and weight bearing index (WBI) were noted in obese subjects. There was a negative correlation between waist hip ratio and aerobic exercise level (oxygen uptake at ventilatory threshold) (r= -0.509, P < 0.01) and also between waist hip ratio and WBI (r= -0.315, P<0.01). Conclusions Lower physical fitness is considered to be a general characteristic of male obese Japanese, and the accumulation of visceral adipose tissue might affect exercise capacity. Chin Med J 2001; 114(7); 707-710

Objective To evaluate the efficacy of allo-UCBT in thalassemia children. Methods Five patients with β-thalassemia major (genotypes were homozygote of β41-42, β654 and double heterozygosities of β41-42/β654, β41-42/-28, β654/-28, respectively) were treated by allo-UCBT. The medican age was 3 year and ten month old (31/2 - 72/12). Four donor-recipient pairs were HLA-identical and one 1 locus mismatch. The patients received a median UCB nucleated cells (NC) of 5.5 × 107/kg body weight [ range (4.7 - 7.7) × 107/kg] and CD34+ CD38- cells 2.8 × 105/kg [range (0.6 - 4.5) × 105/kg] and CFU-GM 1.09 × 105/kg [range (0.24 - 230.00) × lO5/kg]. The conditioning regimen consisted of busalphan 16 - 20 mg/kg, cyclophosphamide 180 - 200 mg/kg) horse antithymocyte globulin (ATG) 90 mg/kg or rabbit anti-lymphocyte globulin (ALG) 25 mg/kg and melphalan of 90 mg/m2 was added to four cases. Cyclosporins A (CsA) alone was administered in 3 cases, and a combination of CsA and methotrexate in the other 2 Cases. Results Four cases were engrafted, but one rejected and recurred thalassemia state at day 60 past UCBT. One case had autologous reconstitution. The reconstitutions were found in all patients with neutrophils 0.5 × 109/L at day 19 (14 - 22), platelets > 20 × 109/L at day 37 (21-63). Two cases developed grade I and Ⅱ acute GVHD respectively. None developed chronic GVHD. Three patients survived disease-freely (survival with exthalassemia state). The median follow-up was 18 months, the probability of survival was 100%. Conclusion Allo-UCBT could reconstitute hematopoiesis with low frequency and mild grade of GVHD. It is an effective treatment for thalassemia. Chin J Hematol 2001; 22: 182-185.

Objective To investigate the effect of intrathecal injection (IT) with methotrexate (MIX) plus dexamethasone (DXM) in treating central nervous system involvement in systemic lupus erythematosus (CNS lupus). Methods Twenty-four CNS lupus patients that were refractory to conventional steroid therapy were selected for IT with MTX 10-20 mg plus DXM 10 - 20 mg. The effects and side effects of IT were closely observed. Results The symptoms and signs of 22/24 (91.7%) CNS lupus patients receiving IT improved considerably. Cerebrospinal fluid pressure, protein and WBC levels declined from 201.5±155.4 mm H2O, 145.2±87.6 mg/dl and 25.1±14.3/mm3 to 128.7 ± 108.1 mm H2O, 60.8 ± 38.3 mg/dl and 6.8 ± 2.1/mm3 respectively. Transient side effects were observed in 4 patients: 1 with itching sensation of lower limbs, 2 with headache and 1 with incontinence. Conclusion IT with MTX plus DXM is a promising method for treating CNS lupus and deserves further investigation. Chin Med J 2001; 114(7):764-766

Objective To study the pathogen of root surface caries in the elderly. Methods Plaque samples from the experimental group and the control group were collected. After culturing in selective and non-selective media, the different bacteria were isolated. The counts and the positive rates of total bacteria, porphyromonas group, pervotella group, streptococcus mutants group, actinomyces group and lactobacillus group were compared. Results The counts of total bacteria, streptococcus mutants, actinomyces and lactobacillus of the experimental group were higher than those of the control group. The positive rates of actinomyces and lactobacillus of the experimental group were significantly higher than those of the control group. Conclusion Actinomyces and lactobacillus are the main related pathogens of root surface caries in the elderly. Chin Med J 2001; 114(7):767-768

Objective To analyze the epidemic trend of the bacteria causing burn wound infection, and to explore the influencing factors. Methods Bacterial ecology on burn wounds was investigated in 210 burn patients admitted to our hospital from January 1999 to December 2000, and the results were compared to those done in the 1970s, 1980s and 1990s. Results Since 1999, G+ cocci increased significantly (52.17%) when compared with those of the past three decades (P < 0.01)> while G- bacilli decreased significantly (46.48%, P < 0.01). The ratio of Pseudomonas aeruginosa in the past two years was evidently lower than those in the early 1970s and 1990s (P < 0.01), while the ratio of staphylococcus aureus was much higher than those in the mid-1980s and 1990s (P < 0.01). Methacillin-resistant Staphylococcus aureus (MRSA) accounted for about 85%, significantly higher than its rate in the mid-1980s and the beginning of the 1990s (P < 0.01), with an increasing trend. Conclusion The increased incidence of Staphylococcus aureus in our unit in the past two years might be closely related to the preferential application of Imipenem in burn infection control. Furthermore, the ratio of MRSA in Staphylococcus aureus could not be lowered by vancomycin. Chin J Burns 2001; 17: 80-82