2005年 · 第118卷第03期

Background The expression of therapeutic gene and its anti-tumor effects will be augmented and a synergism of oncolytic virus with the therapeutic gene is speculated. This study was undertaken to assess the anti-tumor effects of a novel gene-viral therapeutic system CNHK300-mEndostatin (CNHK300-mE) in hepatocellular carcinoma (HCC). Methods A novel gene-viral therapeutic system named CNHK300-mE was constructed using the human telomerase reverse transcriptase (hTERT) promoter to drive the expression of the adenovirus El A gene and cloning the therapeutic gene mouse endostatin into the adenovirus genome. By the tissue culture infectious dose 50 (TCID50) method and cytoviability assay, the replicative and cytolytic capabilities of CNHK300-mE in two HCC lines (HepGII and Hep3B) and one normal cell line (MRC-5) were analyzed, and the transgene expressions of mouse endostatin in vitro and in vivo were detected by Western blotting and ELISA assay. Tumor growth suppression and anti-angiogenesis effects in vivo were investigated using nude mice xenografts model derived from SMMC-7721 HCC cells. Results The 3296-fold replicating capacity of CNHK300-mE in HCC cell lines versus in the normal cell line at 96 hours post infection and the 25 -fold effective dose for killing 50% cells (ED50) in the normal cell line versus HCC cell lines, which were both superior to ONYX-015, were observed. Tumor growth suppression of CNHK300-mE superior to either Ad-mE or ONYX-015 was demonstrated (P <0. 01) and the anti-angiogenic effects in vivo superior to Ad-mE were also observed with immunohistochemical staining of von Willebrand factor. In comparison with non-replicative adenovirus Ad-mE, the transgene expression of mE mediated by CNHK300-mE was significantly higher in vitro (P <0. 005) and in vivo (P <0. 05). Conclusion Being capable of replicating in and lysing the telomerase-positive HCC cells and mediating effective expression of the therapeutic gene in vitro and in vivo, the novel gene-viral therapeutic system CNHK300-mE is potentially effective in the treatment of HCC. Chin Med J 2005; 118(3): 179-185

Background In hypertrophic scar tissue, no sweet gland and hair follicle exist usually because of the dermal and epidermal damage in extensive thermal skin injury, thus imparing regulation of body temperature. This study was designed to reveal the morphological and distributional characteristics of the sweat glands in normal skin and hypertrophic scar obtained from children and adults, and to study the possible interfering effects of the scar on regeneration of the sweat gland after burn injury. Methods Biopsies of hypertrophic scar were taken from four children (4-10 years) and four adults (35 -51 years). Normal, uninjured full-thickness skin adjacent to the scar of each patient was used as control. Keratin 19 (K19) was used as the marker for epidermal stem cells and secretory portion of the sweat glands, and keratin 14 (K14) for the tube portion, respectively. Immunohistochemical and histological evaluations were performed. Results Histological and immunohistochemical staining of skin tissue sections from both the children and adults showed K19 positive cells in the basement membrane of epidermis of normal skin. These cells were seen only single layer and arranged regularly. The secretory or duct portion of the eccrine sweat glands was situated in the dermis and epidermal layer. However, in the scar tissue, K19 positive cells were scant in the basal layer,and the anatomic location of the secretory portion of sweat glands changed. They were located between the border of the scar and reticular layer of the dermis. These secretory portions of sweat glands were expanded and were organized irregularly. But a few K14 positive cells were scattered in the scar tissues in cyclic form. Conclusions There are some residual sweat glands in scar tissues, in which the regeneration process of active sweat glands is present. Possibly the sweat glands could regenerate from adult epidermal stem cells or residual sweat glands in the wound bed after burn injury. Chin Med J 2005; 118(3): 186-191

Background No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1)into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen. Methods Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type I collagen. In addition to hepatic hydroxyproline content,hepatic collagen types I and III were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining. Results Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P <0.01),hepatic hydroxyproline content and the accumulation of collagen types I and III were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P <0. 01) as compared with the model group. Conclusion The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis. Chin Med J 2005; 118(3): 192-197

Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen -activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD. Methods We assessed the effects of NCTD on cell growth inhibition using the 3 -(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected. Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2 - terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression. Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs. Chin Med J 2005; 118(3): 198-203

Background The treatment of hypoparathyroidism (HPT) is still a difficult clinical problem, which necessitates a new therapy. Gene therapy of HPT has been valuable, but how to improve the gene transfer efficiency and expression stability is a problem. This study was designed to optimize the gene therapy of HPT with hematopoietic stem cells (HSCs) recombined with the parathyroid hormone (PTH) gene. Methods The human PTH gene was amplified by polymerase chain reaction (PCR) from pcDNA3. 1 -PTH vectors and inserted into murine stem cell virus (MSCV) vectors with double enzyme digestion (EcoRI and Xhol). The recombinant vectors were transfected into PA317 packaging cell lines by the lipofectin method and screened by G418 selective medium. The condensed recombinant retroviruses were extracted and used to infect HSCs, which were injected into mice suffering from HPT. The change of symptoms and serum levels of PTH and calcium in each group of mice were investigated. Results The human PTH gene was inserted into MSCV vectors successfully and the titres were up to 2 × 107 colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng • 10 6 •cell-1 per 48 hours. The wild type viruses were not detected via PCR amplification, so they were safe for use. The mice suffering from HPT recovered quickly and the serum levels of calcium and PTH remained normal for about three months after the HSCs recombined with PTH were injected into them. The therapeutic effect of this method was better than simple recombinant retroviruses injection. Conclusions The recombinant retroviral vectors MSCV-PTH and the high-titre condensed retroviral solution recombined with the PTH gene are obtained. The recombinant retroviral solution could infect HSCs at a high rate of efficiency. The infected HSCs could cure HPT in mice. This method has provided theoretical evidence for the clinical gene therapy of HPT. Chin Med J 2005; 118(3):204-209

Background Manometric pressure asymmetry of the anal sphincter exists in the anal canal. There are reports about the anatomy of the anal sphincter, but the relationship between the configuration and the pressure asymmetry of the anal sphincter is not clear. This study is to investigate the anatomic evidence and clinical application of anal sphincter pressure asymmetry. Methods PC polygram HR at the state of relaxing and squeezing was used in 27 normal children and 12 abnormal ones with fecal incontinence. Results In normal children, longitudinal pressure gradients existed at eight channels in the anal canal, and the maximal pressure 1 cm from the anal verge. Longitudinal pressure asymmetry changes of eight channels also existed in the anal canal, from 3 cm to 2 cm to 1 cm from the anal verge. The high pressure distribution changed from the posterior to the anterior anal canal. Anteriorly, 1 cm from the anal verge, the maximal pressure was formed in the anal canal. However, neither longitudinal pressure gradients nor longitudinal pressure asymmetry changes were seen in patients with fecal incontinence. Conclusion The configuration and function of the striated muscle complex possibly contribute to the formation of the pressure asymmetry of the anal sphincter, which is essential to anal control. Chin Med J 2005; 118(3):210-214

Background Tuberous sclerosis (TS) is an autosomal dominant disorder with a significant range of clinical expressions. The involvement of vital organs, such as the brain, kidney, heart and lung is the main cause of death in patients with TS. The aim of this study is to summarize the charateristic cutaneous features and common extracutaneous involvement of TS, which are helpful to the early detection of visceral involvement. Methods The analyzed clinical data from 78 patients with TS included those from detailed history, physical and dermatological examination, cranial computed tomography (CT) and magnetic resonance imaging (MRI),abdominal ultrasonography, chest roentgenography, hand and foot X-ray and ophthalmologic examination. Results The skin, brain and kidney were involved frequently in TS patients. Hypomelanotic macules were the most common and earliest cutaneous lesions. Their number was more than 3 in 81. 5%of the patients. They were followed by facial angiofibromas and Shangreen's patch in a decreasing frequency. Forehead plaque, facial angiofibromas and Shagreen's patch appeared in patients at mean age of 2. 6, 6. 0 and 8. 1 years respectively. Cranial CT showed a high positive rate in TS patients. Conclusions Cutaneous features of TS are helpful in the early diagnosis of the disease. Hypomelanotic macules are especially important for patients with epilepsy or babies whose number of hypomelanotic malues is more than 3. Cranial CT is of great value in the diagnosis of TS. The involvement of visceral organs such as the brain and kidney should be examined in TS patients. Chin Med J 2005; 118(3):215-219

Background The second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene,which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells. Methods After the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry. Results Smac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P <0. 01). There was no significant difference in cellular viabilities between them (P> 0. 05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22. 42%(P <0. 01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P <0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P <0. 01), while its activities were increased by 3. 42 times (P <0. 01). Conclusions Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis -inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer. Chin Med J 2005; 118(3):226-230