2005年 · 第118卷第11期

Background Mucosal immunity is important to defense against dental caries. To enhance mucosal immunity, a DNA vaccine mucosal delivery system was prepared by encapsulating anticaries DNA vaccine (plasmid pGJA-P/VAX) in chitosan under optimal conditions and the characteristics of the microparticles was investigated. Furthermore, the release properties and protective action of microparticles for plasmid were studied in vitro. Methods Plasmid loaded chitosan microparticles were prepared by complex coacervation. Three factors, concentration of DNA, sodium sulfate, and the chitosan/DNA ratios in complexes ［better expressed as N/P ratio: the number of poly nitrogen (N) per DNA phosphate (P)］ influencing preparation were optimized by orthogonal test. The characteristics of microparticles were evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). DNA release rate of microparticles in similar gastro fluid (SGF) or similar intestinal fluid (SIF) at 37℃ was determined by ultraviolet spectrophotometry. Results High encapsulation efficiency (96.8%) was obtained with chitosan microparticles made under optimal conditions of 50 mmol/L Na2SO4, 200 μg/ml DNA and N/P ratio of 4. The size of particles was about 4 to 6 μm. The encapsulation process did not destroy the integrity of DNA. When incubated with SIL, after a release of about 10% in the first 60 minutes, no further DNA was released during the following 180 minutes. When incubated with SGL, the microparticles released a small burst (about 11%) in the first 60 minutes, and then slowly released at a constant, but different rate. Conclusions These chitosan microparticles showed suitable characteristics in vitro for mucosal vaccination and are therefore a promising carrier system for DNA vaccine mucosal delivery.

Background The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. Methods Human limbal cells were isolated and cultivated in vitro. Cytokertins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. Results On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days’ labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. Conclusions Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.

Background Since being reclassified by WHO in 1996, solid pseudopapillary tumour (SPT) of pancreas has been recognized as the internationally accepted name. Clinicians are lacking in knowledge of this rare disease so the misdiagnosis and inappropriate therapy are hard to avoid. The clinic data on 22 patients were summarized to study the misdiagnosis and treatment of a sample of SPTs. Methods Twenty-two female patients with SPT were studied retrospectively and divided into two groups, the misdiagnosed group and the correctly diagnosed one. The analyses were performed with Fisher test with accurate probability for categorical data, and Krusdal-Wallis test for ranked data. Results The rate of misdiagnosis in this sample was 45.5%. The misdiagnosed SPTs were apt to be the incomplete capsule ones (P=0.020), which resulted in obvious difficulties during operation (P=0.024). In the misdiagnosed SPT group, the medical expenses increased significantly (P=0.042), and the number of days in hospital greater than in correctly diagnosed group (P=0.041). Conclusions Although SPT has low malignancy with excellent prognosis after surgical treatment in most patients, the misdiagnosis of SPT increases the social and economic burdens on patients. It is important to analyse the causes of misdiagnosis.

Background The association between the infection of Porphyromonas gingivalis, Actinobacillus actinomy-cetemcomitans and Treponema denticola in chronic periodontitis (CP) and the severity of periodontal disease remains to be elucidated. The aim of this study was to investigate the subgingival infection frequencies of three periodontopathic bacteria in Chinese CP patients and to evaluate the correlations between infection by these bacteria and periodontal destruction. Methods A multiple PCR assay using primers derived from 16SrDNA genes of P. gingivalis, A. actinomy-cetemitans and T. denticola was established to measure simultaneously the presence of the three microbes in 162 subgingival samples from 81 Chinese CP patients. Results The positive rates of P. gingivalis, A. actinomycetemitans and T. denticola in the subgingival samples were 84.6%, 83.3% and 88.3%, respectively. Of the subgingival samples, 68% revealed the coinfection of all the three microbes. The infection rates with P. gingivalis, A. actinomycetemitans or T. denticola alone was 5.9% (1/17), 17.6% (3/17) and 76.5% (13/17), respectively. A close association was present between the A. actinomycetemitans infection and gingival index (GI) (P<0.01), but not between P. gingivalis or T. denticola infection and GI (P>0.05). P. gingivalis and A. actinomycetemitans were more frequently detectable in middle and deep pockets than in shallow ones (P<0.01), while T. denticola was found remarkably often in deep pockets (P<0.05). The coinfection rate of the three microbes was significantly higher in sites with severe periodontitis than in those with mild periodontitis (P<0.01). Conclusions The multiple PCR established in this study can be used as a sensitive and specific method to simultaneously detect all three microbes in subgingival samples. A. actinomycetemitans infection may be associated with CP and play an important role in the periodontal tissue destruction. The coinfection of P. gingivalis, A. actinomycetemitans and T. denticola can cause more serious periodontal destruction than infection of any one or two of the three microbes.

Background Regulated on activation, normal T-cell expressed and secreted (RANTES) plays a critical role in T-lymphocyte activation and proliferation. The process is involved in both acute and chronic phases of inflammation. The present study was to ascertain the possible correlations between chronic hepatitis B virus (HBV) infection and the RANTES gene polymorphisms and their expression. Methods The study included 130 HBV negative healthy donors and 152 patients with chronic hepatitis B (CHB) virus infection. The polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) were used to detect RANTES gene single nucleotide polymorphisms (SNPs). RANTES levels in the platelet depleted plasma were detected by enzyme linked immunosorbent assay (ELISA). Results RANTES alleles -403G, -28C and In1.1T were the predominant alleles in the subjects studied. No significant correlation was found between CHB infection and the RANTES alleles, while a significant correlation was found between CHB infection and increased RANTES expression in platelet depleted plasma (P<0.05). Conclusions SNPs in RANTES gene do not affect chronic HBV infection or the outcome of interferon-α treatment in patients positive for HBV “e” antigen (HBeAg+). However, patients with CHB infection express the higher levels of plasma RANTES, which is thus associated with CHB infection.

Background Up to now there have been no satisfactory drugs to treat psychiatric disorders, and now bioactive compound from entomagenous fungi (BCEF0083) is a new type of bioactive compound from entomopathogenic fungi. Our previous investigations have shown that BCEF has an inhibition effect on monoamine oxidase. So, BCEF may be a latent antidepressant. This study aimed at observing the antidepressant effects and its mechanism of BCEF in the chronic unpredictable stress models in mice. Methods The antidepressant effects of BCEF were examined on the chronic unpredictable stress models in mice. Sixty mice were randomly divided to six groups. Animals were housed and isolated except saline group. Mice were exposed to different stressors per day randomly from day 1 to day 21. Body weight were weighed on day 1, day 10 and on day 21 during the 21-day stress procedure. Awarding response was detected by using method of calculating the 24-hour consumption of saccharum water. Step through test was used to evaluate the behavioral response. AVP contents in plasma were also detected by using radioimmunoassays. Results Chronic unpredictable stress resulted in a significant decrease of the body weight and could apparently cause escape behavior disturbance and gradual reduction of sensitivity to reward in animal models. Drug treatment (BCEF 25, 50, 100 mg/kg) could significantly ameliorate the decreased body weight and effectively reverse the escape behavior disturbance. The gradual reduction of sensitivity to reward, the anhedonic state, was also effectively reversed by BCEF. BCEF (50, 100 mg/kg) could also effectively restore the AVP content in the plasma. Conclusions This evidence suggests that BCEF can effectively inhibit the depression behavior and show strong antidepressant effect. BCEF can effectively restore the plasma AVP release and this may be an important mechanism of its antidepressant effect.

Background RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). Methods The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student’s t tests. P<0.05 was considered statistically significant. Results In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P<0.05), 30.1% (P<0.01) (transient transfection) and 37.6 % (P<0.05), 28.0% (P<0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P<0.05), 54-fold (P<0.01) (transient transfection) and to 108-fold (P<0.05), 50-fold (P<0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. Conclusions shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.

Background We randomly sampled a healthy community to evaluate the prevalence and associated risk factors of urinary incontinence. Methods The survey was performed in Fuzhou, China. Of women over twenty years of age in the city, 3.0% were randomly selected and 4684 evaluated by Bristol Female Urinary Tract Symptoms Questionnaire. Results Of the women in Fuzhou, 19.0% had urinary incontinence. The prevalence of stress incontinence, urge incontinence and mixed incontinence was 16.6% (n=777), 10.0% (n=468), 7.7% (n=360) respectively. The prevalence of the three types of urinary incontinence increased significantly with age (P<0.01). In multiple logistic models, age (OR, 1.3, 95%CI, 1.1－1.4), vaginal delivery (3.0, 1.9－4.7), parity>2 (2.1, 1.5－2.9), hypertension (2.7, 1.4－5.6), constipation (2.6, 1.8－3.8), alcohol consumption (4.7, 1.1－20.2), episiotomy (1.7, 1.4－2.0), higher body mass index (BMI, 1.8, 1.5－2.2) and unskilled worker (0.7, 0.5－0.8) were potential risk factors for stress incontinence. Urge incontinence was associated with age (OR, 1.3, 95%CI, 0.9－1.3), menopause (1.6, 1.1－2.4), Caesarean delivery (0.2, 0.1－0.5), parity>2 (2.6, 1.8－3.8), constipation (2.3, 1.4－3.7), foetal birthweight (1.7, 1.1－2.4), episiotomy (1.4, 1.1－1.8), higher BMI (1.5, 1.2－2.0) and unskilled worker (0.7, 0.5－0.9). The prevalence of urinary incontinence and its subtypes in Chinese women is lower than that of occidental women. In China, age, vaginal delivery, parity, hypertension, constipation, alcohol consumption, episiotomy, higher BMI are potential risk factors for stress incontinence. Urge incontinence is associated with age, menopause, Caesarean delivery, parity, constipation, foetal birthweight, episiotomy, higher body mass index.