洛美沙星人工抗原的合成及鉴定

通过化学方法制备了洛美沙星的免疫抗原和包被抗原;分别采用碳二亚胺（EDC）法和氯甲酸乙丁酯法把洛美沙星与BSA进行偶联制备了人工免疫抗原LOM-BSA,EDC法制备了包被抗原LOM-OVA,经非变性PAGE、紫外分光光度法测定了人工合成抗原的偶联比,EDC法和氯甲酸乙丁酯法制得的LOM-BSA两分子结合比率分别为4∶1和11∶1,LOM-OVA中两分子结合比率为12∶1。这为洛美沙星单克隆抗体的制备奠定了基础。     

Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 µg/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

Role of androgen receptor in the pathogenesis of human prostate cancer

AIM: To investigate molecular mechanisms associated with the acquisition of androgen-independent growth of human prostate cancer during progression. METHODS: Upon continuously passaging, the androgen-independent LNCaP cell model was established. The expression of AR and PSA proteins in the course of prostate cancer progression was determined by Western blot. RESULTS: Upon continuous passage, the biological behavior of androgen-dependent parental LNCaP C-33 cells (passage number less than 33) was altered. LNCaP C-81 cells (passage number higher than 80) exhibited more aggressive growth and lower androgen-dependence than C-33 and C-51 cells (passage number between 33 and 80). C-81 cells secreted a higher level of PSA and the degree of DHT stimulation on PSA secretion was lower in C-81 cells than C-33 cells. The three LNCaP subclones expressed a similar level of total AR protein, but C-81 cells showed a characteristic loss of expression of the AR-B and increase in expression of the AR-A. CONCLUSION: Multiple factors, including the different expression of AR isoforms, contribute to the development of androgen-independent growth of prostatic carcinoma cells.     

Changes of serum T-PSA,F-PSA and F/T ratio in patients with prostate cancer (PCa) and its clinical significance

AIM: To evaluate the changes of serum total prostate specific antigen (T-PSA), free prostate specific antigen (F-PSA) and the ratio of F-PSA to T-PSA (F/T) in patients with prostate cancer and its clinical significance. METHODS: The concentrations of T-PSA and F-PSA in serum were measured by micropartical enzyme immunoassay (MEIA) using AxSYM System, and the F/T ratio was calculated. RESULTS: Before operation, the concentrations of T-PSA and F-PSA in patients with PCa were much higher and F/T ratio was significantly lower than that in patients with benign prostate hyperplasia (BPH). T-PSA and F-PSA levels decreased, but F/T ratio increased after operation in PCa and BPH. F/T ratio in 83.5% PCa and 6.5% BPH was less than 0.16. To diagnosis PCa, the sensitivity of F/T ratio was 83.5%, and the specificity was 86.7%. CONCLUSION: Serum T-PSA, F-PSA and F/T ratio are important parameters for the early diagnosis of prostate cancer.     

捻转血矛线虫ZJ株24000分泌排泄(ES)蛋白基因的克隆和序列分析

通过 RT- PCR方法 ,从捻转血矛线虫成虫总 RNA中扩增得到特异性片段 ,并把这一基因片段克隆到 p U C18克隆载体上 ,进行测序及同源性比较。序列分析可知 ,其核苷酸序列与国外已发表的 2 4 0 0 0分泌排泄抗原基因的同源性为 99.2 %。表明本试验成功提取了捻转血矛线虫成虫总 RNA,并用 RT- PCR方法克隆了 2 4 0 0 0分泌排泄抗原基因 ,为该基因的表达及其免疫学作用研究奠定了基础     

Effect of inhaled nitric oxide on adhesion molecule CD11b expression in lung neutrophils of meconium aspiration rabbits

AIM:To evaluate effects of inhaled nitric oxide(iNO) on adhesion molecule CD11b expression on lung neutrophils in experimental meconium aspiration syndrome(MAS) rabbits treated with conventional mechanical ventilation under room air or 100%O₂. METHODS:Animals were randomly allocated to 8 groups(n=48) of 6 each: two MAS model groups(under room air or 100%O₂ without iNO treatment), 6 treatment groups were treated with continuous NO inhalation at a dose of 0.2×10^-6mol/L, 0.33×10^-6mol/L or 0.67×10^-6mol/L respectively for 12 hours under room air or 100%O₂. Mean systemic arterial pressure(SAP) and methemoglobin (MeHb) were performed at basement time, 0, 2, 4, 12 hours. Expression of CD11b on neutrophils in the bronchoalveolar lavage fluid(BALF) was detected with flow cytometry. RESULTS:SAP, MeHb at different time among different groups were within the normal scale. CD11b expression on the neutrophils in the BALF significantly decreased in groups of inhalation 0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO, compared with the two MAS model groups. (x±s: under 21%O₂, 0.33×10^-6 mol/L NO, 121±20 υs 392±204; 0.67×10^-6 mol/L NO, 112±30 υs 392±204;under 100%O₂, 0.33×10^-6 mol/L NO, 113±24υs293±65; 0.67×10^-6 mol/L 102±114 υs293±65, P<0.05). 0.2×10^-6mol/L NO inhalation did no effect on CD11b expression. (x±s:21%O₂, 190±101 υs 392±204; 100%O₂, 222±85 υs 293±65; P>0.05). No statistic difference was observed between groups inhaled 0.33×10^-6 mol/L NO and 0.67×10^-6mol/L NO. CONCLUSION:0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO inhalation down-regulated the CD11b expression on the neutrophils in BALF to reduce the sequestration of neutrophils in rabbit lung.     

Effect of hypoxia on the expression of proliferating cell nuclear antigen and phenotype of cardiac fibroblasts

AIM:To investigate effect of hypoxia on the expression of proliferating cell nuclear antigen(PCNA) and phenotype of cardiac fibroblasts(CFs). METHODS:The purified cardiac fibroblasts were cultured and divided randomly into there groups :control group, moderate hypoxia(MH) group and severe hypoxia(SH) group. After 72 h, MTT method was used to investigate the proliferation of CFs, and the ultrastructure of fibroblasts were observed with transmission electron microscopy. The expression of PCNA and α-actin in cardiac fibroblasts were measured by the means of immunohistochemistry and laser scanning confocal microscopy, respectively. RESULTS: MTT A_{490 nm}value of MH group was significantly higher than that of control group by (18.4±25.0)% (P＜0.05), whereas MTT A_{490 nm} value of MH group was significantly lower than that of control group by (15.8±25.0)% (P＜0.05).The immunohistochemistry experiment showed that there was basal PCNA expression in control CFs, and it was increased in MH CFs(P＜0.05 vs control group), but there was no significant difference between SH and control CFs. Observed with transmission electron microscopy, the control CFs had typical phenotype of fibroblasts: shuttle-shaped, abundant rough endoplasmic reticulums (RER) and golgi complexes, but few mitochondria or micromyofilaments in cytoplasm. After MH for 72 h, there appeared lots of micromyofilaments in cytoplasm, but there were little micromyofilaments in SH CFs. Observed with laser confocal scanning microscopy, there was lower expression of α-actin in CFs of control group, while many regular bundles of micromyofilaments and the expression of α-actin were signifiantly increased (P＜0.05 vs control group) in CFs treated with MH. The α-actin expression in severe hypoxia CFs was not significantly different from control group. CONCLUSION:MH made CFs have the characteristics of myofibroblasts phenotype and enhanced PCNA expression.     

病毒细菌协同凝集试验在蔬菜病毒检测中的应用

在蔬菜病毒病的病毒细菌协同凝集试验（简称ＶＢＡ法）中,首次比较了金黄色葡萄球菌Ｃｏｍｎ１和Ｎｏ．１８００菌株对ＶＢＡ灵敏度的影响,结果表明用Ｎｏ．１８００菌株代替国内、外通用的Ｃｏｍｎ１菌株,ＶＢＡ检测ＴｏＭＶ、ＣＭＶ和ＰＶＸ纯病毒的灵敏度提高了２～１０倍,达３．４～１３．０ｎｇ／ｍＬ,检出病汁液的最大稀释度达１０３～１０５,其灵敏度接近于Ａ蛋白酶联法。在此基础上,又将经抗血清致敏的金黄色葡萄球菌液体免疫剂冷冻干燥,制成一种新型的固体免疫剂,可在４℃下保存;在－１０℃贮藏１年后,用ＶＢＡ法检测ＴｏＭＶ、ＣＭＶ和ＰＶＸ纯病毒的灵敏度分别为８１、２０５和２１０ｎｇ／ｍＬ;即使在１５℃下贮藏１年,灵敏度仍高达２～６μｇ／ｍＬ;在２５℃下贮藏３个月,还有一定的效价。而液体免疫剂不能在１５℃下贮藏;在４℃下贮藏１０个月即失效。因此,固体免疫剂既保持了原液体免疫剂灵敏度高、特异性强、快速简便和经济的特点,又增加了耐贮藏的优点,更加适合在基层单位推广使用。     

免疫鸡MDV感染后羽毛根HVT的分离及HVT免疫对MDV抗原检测的影响

５只ＨＶＴ免疫鸡,实验感染ＭＤＶ后的第１～１０周期间,分别采用初次细胞分离培养物上清中的自由病毒粒子鉴定,ｄｏｔ－ｂｌｏｔＤＮＡ杂交和常规ＡＧＰ试验等方法,对并发感染鸡羽毛根进行了病毒及病毒抗原的动态检测与比较。结果显示,在上述羽毛根中可同时存在ＨＶＴ粒子和ＭＤＶＤＮＡ。ＨＶＴ可分离时间为ＭＤＶ攻毒后１～７周;ＭＤＶＤＮＡ最早检出时间为ＭＤＶ攻毒后的第１８～２２天,并达到其持续性（２２～７０ｄ）的检出率最高峰（１００％）;而ＡＧＰ法对此期间鸡只羽毛根ＭＤＶ抗原的最早检出时间在第２２天,且检出率很低。因此,羽毛病毒抗原ＡＧＰ检测法,对免疫鸡只或鸡群ＭＤＶ感染的诊断仍然是特异的,但较单纯感染的检测,其检出率更低,因而更难以反映出免疫鸡群中ＭＤＶ感染的真实状况。