2003年 · 第116卷第10期

Objective To evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii(T. gondii) surface antigenl(SAG1)and rhoptry protein 1(ROP1)in mice as a hybrid gene. Methods Truncated SAG1 and ROP1 DNA fragments were amplified using polymerase chain reaction(PCR)and inserted into pEGFP-N3 vector to construct recombinant plasmid pSAG1-ROP1. NIH3T3 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10 μg plasmid DNA entrapped in liposome. Four weeks after the final booster injection, blood samples were collected and subjected to enzyme-linked immuno sorbent assay(ELISA)to investigate humoral and cell-mediated immune responses. Reversal transcript-polymerase chain reaction(RT-PCR)was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. Dot-blot hybridization was employed in order to detect whether or not the injected DNA was incorporated into the genomic DNA of the immunized mice. Results Green fluorescence was observed in pSAG1-ROP1-transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1-ROP1 was between 58 kDa and 75 kDa. No expression was observed in blank control plasmid-transfected cells. The sera of immunized mice exhibited antibodies to T. gondii tachyzoites and primarily interferon-y and interlukin-2. RT-PCR showed that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post the first injection. Dot-blot hybridization revealed that the presence of foreign DNA in the splenocytes and peripheral blood leukocytes was transient and that no foreign DNA had inserted into the genomic DNA of mice immunized with pSAG1-ROP1. Conclusions Immunization with a liposome-encapsulated DNA construct encoding the T. gondii SAG1 and ROP1 can induce humoral and cell-mediated immune responses. Chin Med J 2003; 116(10): 1561-1566

Objective To investigate the association between histocompatibility leukocyte antigen(HLA)-DRB1 alleles and alveolar echinococcosis(AE). Methods Thirty-five patients with AE in high prevalence areas in Gansu Province of China were tested for the HLA-DRB1 gene using the polymerase chain reaction with sequence-specific primer(PCR-SSP)technique. The results were compared with those of 104 healthy individuals. Results The frequency of the HLA-DRB1 * 040x gene was 26% in the patient group, which was significantly higher than that in the control group(9. 62%)with a relative risk(RR)of 4. 45(χ2 = 13. 67, P<0. 01), and an etiological fraction(EF)of 0. 20. The frequency of the HLA-DRB1 *0701 allele was significantly lower in the patient group(2. 86%)as compared to the control group(13. 94%; χ2 =6. 67, P <0. 05)with a preventable fraction(PF)of 0. 30. The frequencies of other DRB1 alleles were not significantly different. Conclusion Susceptibility to AE is significantly associated with the HLA-DRB1 * 040x. HLA-DRB1 * 0701 gene might confer protection against AE in humans. Chin Med J 2003; 116(10): 1557-1560

Objective To explore the significance of protein kinase C(PKC)in proliferation and infiltration of eosinophils in nasal polyps. Methods With in situ hybridization and immunohistochemistry staining methods, PKC, pro-apoptotic, and anti-apoptotic gene(Bax, bcl-2)expressions were measured in nasal polyp tissues from 26 patients and inferior turbinate mucosa tissues(ITMTs)from 20 healthy persons. The May-Grünwald-Giemsa(MGG)staining method was used to identify eosinophils. Results In eosinophils, the positive cell expressive rates of Bcl-2 mRNA and its protein were significantly higher in the group with nasal polyps than in the ITMT group(P<0. 01). Although the positive cell expressive rate of Bax mRNA and associated protein were a little higher in the group with nasal polyp tissues than in the ITMT group, the difference was not significant(P>0. 05). There was PKC expression in the eosinophils of 26 cases of nasal polyps, but occasional PKC expression in 7 of 20 ITMT cases. In the two groups, PKC positive cell expression was significantly different, and the expression of PKC and bcl-2 mRNA as well as associated protein in eosinophils of nasal polyps showed a remarkably positive relationship(r1 =0. 0875, r2 =0. 0823, P <0. 01). Conclusions Increased PKC expression in eosinophils of nasal polyp tissues is closely associated with apoptosis inhibition, and it is presumed that eosinophil apoptosis inhibition in nasal polyp tissues is obtained by activation of the PKC signal transduction pathway. Chin Med J 2003; 116(10): 1553-1556

Objective To determine the increase of apoptosis and the decrease of smooth muscle cells(SMCs)density in human abdominal aortic aneurysms(AAA). Methods In situ terminal transferase-mediated dUTP nick end labeling(TUNEL)was employed to detect apoptosis of SMCs in patients with AAA(n = 25)and normal abdominal aortae(n = 10). Positive cells were identified by specific cell marker in combination with immunohistochemistry. Meanwhile SMC counting was performed by anti-a-actin immunohistostaining to compare the SMC density. Results TUNEL staining revealed that there was significantly increased apoptosis in AAAs(average 8. 6%)compared with normal abdominal aortae(average 0. 95%, P < 0. 01). Double staining showed that most of these cells were SMCs. Counting of a-actin positive SMCs revealed that medial SMC density of AAAs(37. 5 ±7. 6 SMCs /HPF)was reduced by 79. 1 % in comparison with that of normal abdominal aortae(179. 2 ±16. 1 SMCs /HPF, P <0. 01). Conclusions Significantly increased SMCs of AAA bear apoptotic markers initiating cell death. Elevated apoptosis may result in a decreased density of SMCs in AAA, which may profoundly influence the development of AAA. Chin Med J 2003; 116(10): 1549-1552

Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Methods The recombinant plasmid pCD-hVEGF165 was mixed with collagen and was implanted in the necrotic femoral head. The expression of vascular endothelial growth factor(VEGF)was examined by RNA dot hybridization and immunohistochemical techniques. Repair of the femoral head was observed by histological and histomorphometric analysis. Results The expression of VEGF was detected in the femoral head transfected with the VEGF gene. The femoral head transfected with the VEGF gene showed a significant increase in angiogenesis 2 and 4 weeks after gene transfection and a significant increase in bone formation 6 and 8 weeks after gene transfection on histomorphometric analysis(P <0. 01). Conclusions Transfection of the VEGF gene enhances bone tissue angiogenesis. Repair of osteonecrosis could be accelerated accordingly, thus providing a potential method for therapy of osteonecrosis. Chin Med J 2003; 116(10): 1544-1548

Objective To evaluate the status of eight RHD specific exons in 131 Han Chinese blood donors who were classified as RhD-negative by serological methods and explore the genomic structure of RHD gene among the Han Chinese. The Rh blood group system has the highest prevalence of polymorphisms among human blood group systems and is clinically significant in transfusion medicine. The Rh antigens are expressed on polypeptides encoded by two highly homologous genes, RHD and RHCE. Recent molecular studies have shown that the RhD-negative trait could be generated by multiple genetic mechanisms and is ethnic group-dependent. Methods The polymerase chain reaction using-sequence specific primers(PCR-SSP)was used to amplify exons 2, 3, 4, 5, 6, 7, 9 and 10 of RHD gene and exons 1, 2 and 5 of RHCE gene, as well as intron 4 in each of them. Results The 131 cases of RhD-negative phenotypes consisted of 60 ccee, 58 Ccee, 5 ccEe, 5 CcEe and 3 CCee. Among them, 83 with the Rh ccee or ccEe phenotypes(63. 4%)lacked the eight RHD exons indicated above, while 26 cases with the Rh Ccee, CCee, CcEe phenotypes(19. 9%)had all the RHD exons examined. Twenty-two individuals with the Ccee, CCee, CcEe phenotypes(16. 8%)carried at least one RHD exon. The phenotypes of the RhD negative individuals carrying the RHD gene were Rh CC or Cc, but not cc. Conclusions Three classes of RhD-negative polymorphisms among a population of Han Chinese were observed. Antigen association analysis suggested the existence of a novel class of RhD-negative associated haplotype in Han Chinese. This haplotype consisted of a normal RHCE allele and a nonfunctional RHD gene. It may be beneficial to redefine the RhD-negative blood group among Chinese populations upon clarification of the mechanisms of RHD gene expression and RhD antigen immunization. Chin Med J 2003; 116(10): 1539-1543

Objective To screen the point mutation of the low-density lipoprotein receptor(LDL-R)gene in Chinese familial hypercholesterolemia(FH)patients, characterize the relationship between the genotype and the phenotype and discuss the molecular pathological mechanism of FH. Methods A patient with clinical phenotype of homozygous FH and her parents were investigated for mutations in the promoter and all eighteen exons of the LDL-R gene. Screening was carried out using Touch-down PCR and direct DNA sequencing; multiple alignment analysis by DNASIS 2. 5 was used to find base alteration, and the LDL-R gene mutation database was searched to identify the alteration. In addition, the apolipoprotein B gene(apo B)was screened for known mutations(R3500Q)that cause familial defective apo B100(FDB)by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Results Two new heterozygous mutations in exons 4 and 9 of the LDL-R gene were identified in the proband(C122Y and T383I)as well as her parents. Both of the mutations have not been published in the LDL-R gene mutation database. No mutation of apo B100(R3500Q)was observed. Conclusion Two new mutations(C112Y and T383I)were found in the LDL-R gene, which may result in FH and may be particularly pathogenetic genotypes in Chinese people. Chin Med J 2003; 116(10): 1535-1538

Objective To investigate the effects of magnesium sulfate on neuron apoptosis and the expressions of caspase-3, bax and bcl-2 after cerebral ischemia-reperfusion injury. Methods Thirty-six gerbils were randomly divided into three groups: Sham-operation group(So, n = 12), ischemia-reperfusion group(l-R, n = 12)and magnesium sulfate group(Ms, n = 12). The neuron apoptosis was detected by the terminal deoxynucleotidyl transferase(TdT)-mediated dUTP-fluorescence nick end labeling(TUNEL stain), and the protein expressions of caspase-3, bax and bcl-2 were detected by immunohistochemisty stain. Results Apoptotic neurons and the expressions of caspase-3 and bax were significantly increased in I-R and Ms groups, compared with those in So group. Apoptotic neurons and the expressions of bax and caspase-3 in Ms group were significantly less than those in l-R group. There was no significant difference in the expression of bcl-2 among three groups. Conclusions Magnesium sulfate decreases neuron apoptosis after cerebral ischemia-reperfusion injury, which may be related with the suppressed expressions of caspase-3 and bax. Chin Med J 2003; 116(10): 1532-1534

Objective To investigate clinical features of pheochromocytoma and summarize experiences of anesthetic management during the perioperative period. Methods Two hundred and fifty eight patients who were diagnosed with pheochromocytoma in our hospital were reviewed retrospectively for clinical features. According to different preoperative pharmalogical preparations, perioperative mortalities were analyzed in three periods(Period 1: January 1955-December 1975; Period 2: January 1976-December 1994; Period 3: January 1995-July 2001). In Period 3, hemodynamic changes in the patients undergoing different anesthetic methods were analyzed. Results About 5. 8%(15/258)of pheochromocytoma was an integral part of multiple endocrine neoplasia(MEN)type Ⅱ or mixed type. Sixty percent(149/249)of the patients who had undergone surgery possessed evidence of catecholamine cardiac toxicity preoperatively. Impaired glucose tolerance was found in 59%(147/249)of the patients before surgery. Perioperative mortality was significantly decreased from 8%(5/60)in Period 1 to 1. 2%(1 /75)in Period 2(P <0. 01). No perioperative deaths occurred in Period 3. The volume infused during the operation was significantly higher both in the epidural anesthesia group(3474 ml ±624 ml, P < 0. 01)and in the epidural plus general anesthesia group(3654 ml ± 475 ml, P < 0. 01)than in the general anesthesia group(2534 ml ±512 ml). There were favorable hemodynamic characteristics in patients before removal of the tumor in the epidural anesthesia group and in the epidural plus general anesthesia group, as compared with the general anesthesia group. Conclusions A positive surgical outcome of the excision of pheochromocytoma depends on multiple factors, including careful assessment of potential vital organ damage before surgery and restoration of blood volume by establishing a-blockade preoperatively, meticulous anesthetic management of patients during surgery, and appropriate circulatory support after surgery. Chin Med J 2003; 116(10): 1527-1531

Objective To evaluate the efficacy, safety and stability of posterior chamber phakic intraocular lens(PIOLs)implantation for the correction of high myopia. Methods Thirty-nine eyes of twenty patients with high myopia(between-11.75 and-25. 75 diopters)had a posterior chamber PIOL(Staar ICL)implanted. During 6-48 months' follow-up, visual acuity, refraction, intraocular pressure(IOP), corneal reaction and space between crystal lens and intraocular lens(lOLs)were tested. Results Successful implantation was achieved in all patients. Visual acuity without correction greater than 0.5 was found in 34 eyes at 1 day and 3 months postoperatively. Thirty-five eyes maintained a low negative power of refraction(-1.42 ±1. 32 doipters), which did not prevent the patients from most of their daily activities. During 3-48 months' follow-up, refraction was stable and no cornea edema and glaucoma was found. Two eyes of one patient had corticosteroid glaucoma and another eye showed cataractogenesis under anterior capsular membrane. Conclusion Posterior chamber PIOL implantation is predictable, safe, and effective in the correction of high myopia, and its indications should be carefully selected. Chin Med J 2003; 116(10): 1523-1526

Objective To investigate the K-ras mutation pattern in six pancreatic cancer cell lines from Chinese patients. Methods All six cell lines were analyzed for mutations in exon 1 of the K-ras gene by polymerase chain reaction(PCR)and direct sequencing. Results All 6 pancreatic cancer cell lines had GGT→GTT mutations at K-ras codon 12 but no mutations at codon 13. Conclusion The unique GGT→GTT mutation at codon 12 plays a potential role in the carcinogenesis of pancreatic cancers in Chinese. Chin Med J 2003; 116(10): 1585-1587

Objective To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line, LoVo. Methods A retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8. 2 × 105 CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo. Results After transfection and superinfection, amphotropic retrovirus was collected, and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited endothelial cell growth up to 67%(P < 0. 001), compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group, accompanied by a reduction in vessels, compared with the control group(P <0. 01). Conclusion Retroviruses can allow functional expression of the endostatin gene in human colon tumors, showing promise for an antitumor strategy using antiangiogenesis. Chin Med J 2003; 116(10): 1582-1584

Objective To determine whether the clinical and pathologic characteristics and prognoses of myasthenia gravis(MG)patients below 15 years differ from those patients over 50 years after thymectomy. Methods We reviewed the registry material of 30 pediatric and 32 elderly MG patients after thymectomy, including their age, sex, clinical classification, pathological types, and prognoses. The Chi-square test or Wilcoxon's rank-sum test was used to determine the statistical differences between the children and elderly groups. Results No significant difference was seen in sex distribution between the two groups(Chi-square test, P = 0. 625), but there were differences in clinical classification: more type I was observed in the pediatric group than in the elderly group, but more type Ⅱ or Ⅲ was seen in the elderly group(Wilcoxon's rank-sum test, P < 0. 001). As to pathological types, the pediatric group was also significantly different from the elderly group(Chi-square test, P<0. 01). All of the patients(100%)in the pediatric group had thymus hyperplasia, but in the elderly group more than half(56. 26%)were found to have thymoma(benign or malignant). The prognoses after thymectomy were better in the pediatric group than in the elderly group(Wilcoxon's rank-sum test, P <0. 001). Conclusions Because the prognoses are generally better than those of the elderly patients, we should be careful when operating on pediatric patients of ocular type. The elderly patients tend to receive more aggressive treatment because of more severe generalized types often associated with thymoma and poor prognoses. Both pediatric and elderly patients are seldom associated with other autoimmune disease. Chin Med J 2003; 116(10): 1578-1581