2004年 · 第117卷第09期

Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hN GFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption. Methods rAAV-2 containing hN GFβ gene was constructed. The ability of hN GFβ gene mediated by rAAV-2 vector (rAAV-2/ hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/ hN GFβ. rAAV-2/ hN GFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hN GFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope. Results After 48 hours, hN GFβ content in supernatant was up to (188. 0 ±28. 6) pg/ ml when BHK-21 cells were infected by rAAV-2/ hN GFβ at multiplicity of infection (MOI) 1. 0 ×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hN GFβ. Whole brain hN GFβ content in rAAV-2/ hN GFβ transferred group was up to (636. 2 ±140. 6) pg/ ml. hN GFβ content of BBB disruption in rAAV-2/ hN GFβ infused group increased significantly compared to the control group (P < 0. 05). GFP expression was clearly observed in brain sections of rAAV-2/ GFP transferred group. Conclusion rAAV-2/ hN GFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol. Chin Med J 2004; 117(9):1370-1376

Background This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietn-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector mediated human ANGPTL4 gene transfection. Methods ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and Hep G2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in Hep G2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of Hep G2-ANGPTL4 cells in vivo and in vitro, respectively. Results The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4 ×106 infective viral grains/ ml, and the rate of Hep G2-ANGPTL4 cells expressing GFP was 68.45 %, with an average intensity of fluorescence 31. 67 times greater in Hep G2-ANGPTL4 cells than in Hep G2 cells. The expression of ANGPTL4 mRNA in Hep G2-ANGPTL4 cells was higher than in Hep G2-pMSCV cells (154 % higher) or Hep G2 cells (161 % higher). The proliferation rate of Hep G2-ANGPTL4 cells in vitro was obviously lower than those of Hep G2-pMSCV cells and Hep G2 cells (P <0. 01). The mean volume and weight of tumors seeded from Hep G2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from Hep G2 cells and Hep G2-pMSCV cells (P < 0. 01). Conclusion A stable ANGPTL4-transfected human liver cancer cell line Hep G2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy. Chin Med J 2004; 117(9):1364-1369

Background Multidrug resistance is associated with a poor prognosis in various human cancers. However, the clinical significance of the expression of multidrug resistance-related markers in neuroblastoma is still on debate. In this study, the effect of the expression of p-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance protein (LRP) in neuroblastoma was evaluated. Methods The streptavidin-biotin immunoperoxidase (SP) technique was used to evaluate the expression of P-gp, MRP, and LRP in 70 cases of untreated primary neuroblastoma. Results The frequencies of the expression of P-gp, MRP, and LRP were 61.4 %, 38. 6 %, and 24.3 %, respectively. A significant positive correlation was observed between P-gp and MRP expression (P = 0. 001), as well as between LRP and MRP expression (P = 0. 01). The rates of expression of P-gp and MRP were higher in tumors from patients aged greater than one year old than in tumors from patients aged less than 1 year old at time of diagnosis (P = 0. 01 and 0. 018, respectively). MRP expression in tumors that had metastasized was significantly more frequent than in tumors that had not metastasized (P = 0. 015). The expression of all tested proteins showed a significant relationship with whether or not the tumor had differentiated (P = 0. 006, 0. 000 or 0. 001, respectively). MRP expression was significantly associated with a reduction in both median survival time and 2-year cumulative survival (P = 0. 02). By contrast, P-gp and MRP expression did not correlate with survival. According to Cox regression analysis, only the co-expression of P-gp and MRP had significant prognostic value (relative hazard, 3.513, P = 0. 033). Conclusions The intrinsic, multidrug resistance of neuroblastoma involves the combined effects of P-gp, MRP, and LRP. MRP expression may be an important factor determining prognosis in neuroblastoma. Chin Med J 2004; 117(9):1359-1363

Background p27 is an essential mediator of cell cycle control, which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines. Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/ PI, and DNA ladder electrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40. 3 % and 32. 0 %, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/ PI- apoptotic cell rates in HL-60 and Raji cell lines were 46. 9 % and 35. 7 %, respectively, significantly higher than in the control groups (4. 7 %and 5. 6 %, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma. Chin Med J 2004; 117(9):1353-1358

Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdrl cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method. Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21 % - 37 % of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22. 3 - 45. 8 times, and that to colchicines to 6. 7-11. 35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered. Conclusions CIK cells were successfully transfected with mdrl cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells. Chin Med J 2004; 117(9):1348-1352

Background Many researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains. Methods After ICH induction by stereotactical injection of collagenase type Ⅶ into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU. Results Nestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisoned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Postive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P<0.01), peaked at day 14(P<0.05), and reduced significantly by day 28(P<0.01). Conclusion Endogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains. Chin Med J 2004; 117(9):1342-1347

Background Methylprednisolone (MP), a synthetic glucocorticosteroid, has been broadly studied in experiments on endotoxin-induced shock and septic shock. This study was designed to ascertain whether glycine and MP can protect against organ injury and death caused by hemorrhagic shock, and to elucidate the underlying mechanisms of these protective effects in rats. Method To establish a shock model, Wistar rats were bled to maintain mean arterial pressure at 30 - 50 mmHg for 1 hour and subsequently resuscitated with the shed blood and normal saline. Just prior to resuscitation, the rats were randomly assigned to four groups: sham group (operation performed without inducing shock), shock group, shock + glycine group (glycine injected at the beginning of resuscitation) and shock + MP group (MP injected at the beginning of resuscitation). Results ① Seventy-two hours after resuscitation, the survival rate of rats from the shock group had decreased to 20 %, while the survival rates of rats from the shock + glycine and shock + MP groups were 77. 8 % and 80 %, respectively. The difference was significant (P < 0. 05). ② Eighteen hours after resuscitation, pathological alterations in the organs of the rats were apparent. In rats from the shock group, edema, interstitial leukocyte infiltration, and cellular degeneration occurred in the liver, lungs, kidneys, and heart. Glycine and MP reduced these pathological changes significantly. ③ Eighteen hours after resuscitation, the levels of creatine phosphokinase, transaminases, and creatine were elevated significantly in rats from the shock group, indicating injury to the heart, liver, and kidneys, while these levels were elevated only slightly in the shock + glycine and shock + MP groups. The differences were significant (P < 0. 01). ④There were significant increases in intracellular calcium and production of tumor necrosis factor (INF-α) by isolated Kupffer cells stimulated by endotoxin after hemorrhagic shock. These changes were completely prevented by glycine and MP (P <0. 01). Conclusion Glycine and MP reduce organ injury and mortality caused by hemorrhagic shock by preventing increase of intracellular calcium levels in Kupffer cell, suppressing Kupffer cell activation, decreasing the production of TNF-α by Kupffer cells, and blocking systemic inflammatory responses. Chin Med J 2004; 117(9):1334-1341

Background China is one of the countries with the highest incidence of H. pylori and more than 9090 isolates possessed the cagA gene. This study was to evaluate the biological activity of the H. pylori virulence factor cagA isolated from Chinese patients. Methods cagA DNA fragments were amplified from the genomic DNA and subsequently cloned into the mammalian expression vector for cell transfection and DNA sequencing. cagA protein, phosphorylated-tyrosine cagA and the complex of cagA precipitated with SHP-2 were identified respectively by western blot in the crude cell lysate from conditionally immortalized gastric epithelial cells at 48 hours after transfection with cagA DNA. In addition, the ability of induction of scattering phenotype was examined after transient expression of cagA in AGS cells. Results The C-terminal half of cagA contained only one repeated sequence and three tandem five-aminoacid motifs glutamic acid-proline-isoleucine-tyrosine-alanine (EPIYA). Moreover, the amino acid sequence of D2 region in repeated sequence was aspartic acid-phenylanaline-aspartic acid (D-F-D) which was significantly distinguished from the three repeated sequences and aspartic acid-aspartic adid-leucine (D-D-L) in the western standard strain NCTC11637. Western blot revealed that cagA became phosphorylated in tyrosine site and bound with SHP-2 after transient expression of cagA DNA in gastric epithelial cells. Transient expression of cagA in AGS cells showed that cagA was able to induce the elongation phenotype although to a lesser extent than western strains. Conclusions cagA perturbs cell signaling pathways by binding with SHP-2. However, significant difference exists in amino acid sequence and biological function of cagA in Chinese compared with those of western countries. Chin Med J 2004; 117(9):1330-1333

Background Recently congenital infection with Schistosoma japonicum (S. japonicum) has been demonstrated in pigs, rabbits, mice and dogs. We explored the rabbit as an animal model for the congenital infection of schistosomiasis japonica and assessed the effect of a congenital S. japonicum infection on the resistance of rabbit kittens to a postnatal challenge infection. Methods Sixteen pregnant New Zealand white rabbits were infected with a single dose of S. japonicum cercariae. The exposed animals were divided into three groups according to the gestation age at the time of infection. Diagnosis of prenatally acquired S. japonicum infection in the rabbit kittens was primarily based on serological tests in combination with parasitological and histopathological findings. Congenitally infected kittens were challenged percutaneously with 100 S. japonicum cercariae to assess the effect of a congenital S. japonicum infection on kitten resistance to a postnatal challenge infection. Results The overall prevalence of congenital infection in off spring of infected mothers was 20 % (12/ 60). The congenital infection rate in group L (late gestation) was much higher than in group E (early gestation) and group M (mid-gestation) (P < 0. 05). After a postnatal challenge infection, prenatally infected kittens had a 54. 66 % worm reduction rate, 41. 45 % egg reduction rate, and 51. 76 % granuloma size reduction rate compared to naïve kittens. Conclusions This study demonstrates the possibility of congenital infection of S. japonicum in rabbits and the resistance of congenitally infected kittens to a postnatal challenge infection. These results have important implications not only for epidemiological investigations, but also in designing government control programs for schistosomiasis. Chin Med J 2004; 117(9):1326-1329

Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱbeta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8 %, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24. 921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E. coli JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E. coli. Chin Med J 2004; 117(9):1321-1325

Objective To review the recent developments in and research into binding receptors of hepatitis C virus (HCV) and especially the role of dendritic cell-specitic adhesion receptor (DC-SIGN) in HCV. Data sources Both Chinese- and English-languge literature was searched using MEDLINE (2000-2003) and the databank of Chinese-language literature (2000-2003). Study selection Relevant articles on DC-SIGN and HCV binding receptors in recent domestic and foreign literature were selected. Data extraction Data were mainly extracted from 40 articles which are listed in the references section of this review. Results DC-SIGN, a dendritic cell-specific adhesion receptor and a type Ⅱ transmembrane mannose-binding C-type lectin, is very important in the function of dendritic cells (DC), both in mediating naïve T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by HCV and other viral and bacterial pathogens including human immunodeficiency virus (HIV), Ebola virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropriate entry receptors. Recent report showed that DC-SIGN not only plays a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental to the interaction of DC with T cells during antigen presentation. Conclusions DC-SIGNs are high-affinity binding receptors for HCV. The clinical strategies that target DC-SIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations. Chin Med J 2004; 117(9):1395-1400

Background Corneal dystrophy is a group of inherited blinding diseases of the cornea. This study was to identify the mutations of the keratoepithelin (KE) gene for proper diagnosis of corneal dystrophy. Methods Three families with corneal dystrophy were analysed. Thirteen individuals at risk for corneal dystrophy in family A, the proband and her son in family B, and the proband in family C were examined after their blood samples were obtained. Mutation screening of human transforming growth factor β-induced gene (BIGH3 gene) was performed. Results Five individuals in family A were found by clinical evaluation to be affected with granular corneal dystrophy and carried the BIGH3 mutation W555R. However, both probands in families B and C, also diagnosed with granular corneal dystrophy, harboured the BIGH3 mutation R124H. Conclusion Molecular genetic analysis can improve accurate diagnosis of corneal dystrophy. Chin Med J 2004; 117(9):1418-1421