2005年 · 第118卷第04期

Background The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV)includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix,indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection. Methods The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over -expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles. Results We expressed and purified soluble recombinant protein X4 from E. coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6, cells It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays. Conclusion The results provide the evidence of protein X4 expression following SARS-CoV infection,and may facilitate further investigation of the immunopathological mechanism of SARS. Chin Med J 2005; 118 (4): 267-274

Background There are no reports on exnografting cultured human fetal neocortical cells in this infracted cavities of adult rat brains. This study was undertaken to observe whether cultured human cortical neurons and astrocytes can survive and grow in the infarcted cavities of adult rat brains and whether they interconnect with host brains. Methods The right middle cerebral artery was ligated distal to the striatal branches in 16 adult stroke-prone renovascular hypertensive rats. One week later, cultured cells from human embryonic cerebral cortexes were stereotaxically transferred to the infarcted cavity of 11 rats. The other 5 rats receiving sham transplants served as controls. For immunosuppression, all transplanted rats received intraperitoneal injection of cyclosporine A daily starting on the day of grafting. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin,neurofilament, and microtubule associated protein-2 (MAP-2) was performed on brain sections perfused in situ 8 weeks after transplantation. Results Grafts in the infarcted cavities of 6 of 10 surviving rats consisted of bands of neurons with an immature appearance, bundles of fibers, and GFAP-immunopositive astrocytes, which were unevenly distributed. The grafts were rich in synaptophysin, neurofilament, and MAP2-positive neurons with long processes. The graft/host border was diffuse with dendrites apparently bridging over to the host brain, into which neurofilament immunopositive fibers protruded. Conclusion Cultured human fetal brain cells can survive and grow in the infarcted cavities of immunodepressed rats and integrate with the host brain. Chin Med J 2005; 118(4):275-280

Background Tissue-engineering techniques combined with gene therapy have been recently reported to improve osteogenesis. In this study, tissue-engineered bone constructed by human Bone Morphogenetic Protein 4(hBMP-4) gene-modified bone marrow stromal cells (bMSCs) was explored in an ectopic bone formation model in rabbits. Methods A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected in vitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the target gene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted to explore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined with Natural Non-organic Bone (NNB), a porous hydroxyapatite implant with a dimension of 6 mm × 6 mm × 3 mm, at a concentration of 5 × 107 cells/ml. They were subsequently implanted into 6 rabbits subcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation. Results pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, gene transfer efficiency reached more than 30%. Target gene transfer could strengthen the transcription of BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In the ectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in the bMSCs group was (17. 2 ± 7.1) %, and pEGFP group was (14. 7 ± 6. 1)%, while pEGFP-hBMP-4 group was (29. 5 ±8. 2)%, which was the highest among the groups (F=7. 295, P <0. 01). Conclusions The mammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transfer efficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoted differentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection of bMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bone tissue engineering applications. Chin Med J 2005; 118(4):281-288

Background Bone marrow transplantation (BMT) conditioning procedure is considered as the cause of damage to bone marrow microvasculature and the delay of hematopoiesis recovery. However, hematopoiesis regulation post BMT by vascular endothelial growth factor (VEGF) has not yet been studied. In this study,adenovirus were used to investigate the effects of VEGF gene transfer on preventing damages to bone marrow microenvironment and its promotion of hematopoiesis in post-BMT mice. Methods Recombinant adenovirus (Ad) -enhanced green fluorescent protein (EGFP)/hVEGF165 was injected via tail vein into BALB/c mice undergoing syngeneic BMT. During the different phases post BMT, the distribution of adenovirus and the plasma levels of hVEGF were measured as well as the numbers of white blood cells (WBC), platelet (PLT) and red blood cells (RBC) in peripheral blood. At the same time, the mice were injected with Chinese ink via tail vein, following which the tibias were separated and were used for analysis of bone marrow microvasculature surface area and cellularity. Results Significant expression of EGFP and hVEGF was observed in multiple organs at different phases post BMT, and the plasma level of hVEGF was up to (866. 67 ±97. 13) pg/ml. The recovery of WBC, PLT and RBC of the group treated with recombinant adenovirus Ad-EGFP/hVEGF165 were significantly more rapid than those of other BMT groups (P <0. 05, respectively). At the 20th day post BMT, the percentage of bone marrow microvasculature surface area in group treated with VEGF [(61. 2 ±4. 0)%] returned to normal level [(62. 0 ±5.0)%, P> 0. 05]. The restoration of hematopoiesis was retarded more than that of microvasculature. The cellularity of bone marrow in each group was still lower than that of normal control [(62. 3 ± 4. 0) %, P <0. 05]at the 30th day post BMT, but the percentage in group treated with VEGF at the 20th and 30th days post BMT[(46. 5 ±5. 0)% and (55. 1 ±4. 5)%] exceeded those of other BMT groups (P <0. 05, respectively). Conclusion VEGF gene transfer mediated by adenovirus may protect the hematopoietic microenvironment to promote the restoration of hematopoiesis in post-BMT mice. Chin Med J 2005; 118(4):289-295

Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon (IFN) -γ- endostatin and studied the anti-tumor effect of pEgr-IFN-γ-endostatin gene -radiotherapy in mice bearing Lewis lung carcinoma and its mechanism. Methods Gene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-γ-endostatin, and single-gene expression plasmid pEgr-IFN-γ and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor(TNF)-α secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation. Results The tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-α secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-γgene-radiotherapy group. Conclusion The anti-tumor effect of dual-gene-radiotherapy was significantly better than that of single-gene-radiotherapy by combining the enhancement of anti-tumor immunologic function induced by IFN-γ with the anti-angiogenesis function of endostatin. Chin Med J 2005; 118(4):296-301

Background Mutations in PAX6 gene have been shown to be the genetic cause of aniridia, which is a severe panocular eye disease characterised by iris hypoplasia. However, there is no study to do genetic analysis of aniridia, although there are several case reports in China. Here, we describe a mutation analysis of PAX6 in a large Chinese family with aniridia. Methods Genomic DNA from venous blood samples was prepared. Haplotype analysis was performed with two genetic markers (D11S904 and Dll S935) . Fourteen exons of the PAX6 gene were amplified from genomic DNA. Polymerase chain reaction (PCR) products of each exon were analysed by single strand conformational polymorphism (SSCP). The PCR products having an abnormal pattern were sequenced to confirm the mutation. Results Significant evidence for allele sharing in affected patients was detected suggesting that PAX6 mutation links to aniridia in this family. An extra band corresponding to exon 9 in PAX6 was found by single strand conformational polymorphism analysis in all the aniridia patients in this family, but not detected in the unaffected members. A mutation of C to T was detected by sequencing at the nucleotide 1080 that converts the Arg codon(CGA) to the termination codon (TGA). Conclusions Aniridia is caused by a nonsense mutation of PAX6 gene in the large Chinese kindred. Genetic test is important to prevent the transmission of aniridia to their offsprings in the kindred by prenatal diagnosis. Chin Med J 2005; 118(4):302-306

Background Oncolytic herpes simplex virus (HSV) vectors can be used for cancer therapy as direct cytotoxic agents, inducers of anti-tumor immune responses, and as expressers of anti-cancer genes. In this study, the efficacy of HSV vectors, G47Δ and NV1023 were examined for the treatment of the human breast cancer. Methods Human breast cancer MDA-MB-435 cells were cultured or implanted subcutaneously in BALB/c nude mice. The cells or tumors were inoculated with G47Δ or NV1023, and cell killing or inhibition of tumor growth determined. Both viruses contained the LacZ gene and expression in infected cells was detected with X-gal histochemistry. Results G47Δ and NV1023 were highly cytotoxic to MDA-MB-435 cells in vitro at very low multiplicities of infection. X-gal staining of infected tumor cells in vitro and in vivo illustrated the replication and spread of both viruses. G47Δ and NV1023 inoculation inhibited tumor growth and prolonged mouse survival. Both vectors behaved similarly. Conclusions Oncolytic HSV vectors, G47Δ and NV1023, were extremely effective at killing human breast cancer cells in vitro and in tumor xenografts in vivo. This novel form of cancer therapy warrants further investigation and consideration of clinical application. Chin Med J 2005; 118(4):307-312

Background The heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus. Methods BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleocapsid protein alone or in combination with a plasmid expressing murine interleukin-12(pcIL-12). Booster immunizations were employed 2 times at 2 - week interval. To evaluate the humoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-γ in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization. Results Antigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1: 50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3. 1 + S alone, in which the mean titre was about 1: 70. Interferon-γ was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3. 1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 co-injection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation. Conclusion Humoral and cytokine responses elicited by pcDNA3. 1 + S inoculation can be modulated by coinoculation with pcIL-12 and efficiently induced Thl -dominant immune responses. Chin Med J 2005; 118(4):313-319

Background It has been demonstrated that bone tunnel widening could appear after knee anterior cruciate ligament (ACL) reconstruction, especially for those patients whose ruptured ACL were reconstructed with semitendinosus and gracilis tendons. Many factors can influence the extent of tunnel widening. Few studies have investigated the relationship between bone tunnel widening and rehabilitation procedures. This research was carried out to find the rehabilitation procedures' influence on the tibial bone tunnel widening after ACL reconstruction. Methods Sixty-five cases, whose ACL reconstructions were done using quadrupled semitendinosus and gracilis tendons, were divided into two groups. Group A had 33 cases, 19 men, 14 women, averaged (31. 2 ±12. 4)years old, only ACL reconstruction was done using Paessler's technique, and aggressive rehabilitation procedure was used for function recovery post operation. Group B had 32 cases, 20 men, 12 women, averaged (30. 3 ±10. 3) years old. Except for ACL reconstruction, every patient in group B accepted meniscus repair using re-fixation methods or cartilage repair using microfracture technique, conservative rehabilitation procedure was used post operation. Six months post operation, standard posterior-anterior radiographic plates were taken for each case, CorelDRAW 8. 0 software was used to digitize all X-ray plates and measure the upper, middle and lower parts of the tibial tunnel. Magnification effect of X-ray plates was taken out after measurement. Results Six months after ACL reconstruction the tibial tunnel widening of the upper, middle and lower parts on both the posterior-anterior and lateral X-ray plates in Group A with aggressive rehabilitation procedure was much more serious than in Group B with conservative rehabilitation. KT-1000 knee stability measurement and clinical manifestation showed no difference between the two groups. Conclusions Rehabilitation procedure after ACL reconstruction is one of the reasons for tunnel widening. It not only can directly influence the function recovery of ACL reconstructed knee, but also perhaps indirectly influence the function recovery and long-term clinical result of the operated knee by influencing the tunnel widening. Chin Med J 2005; 118(4):320-326