苹果花脸病田间病情发展及ASSVd在组培条件下的传播

为了明确苹果锈果类病毒（apple scar skin viroid，ASSVd）引起的苹果花脸病在田间的病情发展情况以及ASSVd在组培条件下的传播方式，2012—2015年对河北省顺平南神南村3个果园进行持续调查和检测。结果显示，果园A、B、C的显症率分别从3.00%、19.35%和10.00%上升至10.00%、34.41%和22.00%，带毒率分别从11.00%、20.43%和14.00%上升至23.00%、37.63%和30.00%，带毒率和显症率都逐年增加，而且带毒率大于显症率，即有些带毒果树不显症。发病果树田间分布有明显的发病中心。通过高通量测序发现显症果树ASSVd vsiRNA reads数是带毒未显症reads数的4.08倍，同时实时荧光定量PCR检测发现显症树ASSVd病毒积累量显著高于带毒未显症树。以携带ASSVd的组培苗和无毒组培苗为试材，利用RT-PCR检测明确了ASSVd可通过伤口沾染带毒汁液、组培剪污染、含毒培养基、根系接触进行传播，其中根系接触传染率较高。     

苹果锈果类病毒山东栖霞分离物的分子鉴定及序列分析

本研究以采自山东省栖霞市的两个苹果样品为试验材料,利用二维电泳、斑点杂交、RT PCR等分子生物学技术对样品中的苹果锈果类病毒(ASSVd)带毒情况进行了检测。结果表明：两个样品均携带ASSVd。对两个样品中的ASSVd进行克隆测序,并利用生物学软件DNAMAN对所得序列进行分析,结果表明：与世界上首次报道的ASSVd序列(X17696)相比,本研究所得到的ASSVd序列变异较小,相对比较保守。     

Ectopic pregnancy through a caesarean scar in a ewe

CASE HISTORY: A routine ultrasonographic examination for pregnancy diagnosis in a 3-year-old ewe revealed a normal embryo located in the uterine cavity and an abnormal mass located in the abdomen. At the time of examination, the ewe was 4.5?months pregnant, but 10?months previously she had undergone a caesarean section due to dystocia.

CLINICAL FINDINGS: The abnormal mass (12?cm × 8?cm) was located outside the uterine cavity; it was anechoic at the periphery, and in the centre there was an echogenic embryo-like structure. Exploratory laparotomy was performed, which revealed an ectopic pregnancy through the caesarean scar.

TREATMENT: The ectopic foetus was excised and the caesarean scar was repaired. The ewe made a complete recovery and the intrauterine pregnancy was completed to full-term, resulting in the birth of a healthy female lamb 35?days after the removal of the ectopic foetus.

CLINICAL RELEVANCE: Ectopic pregnancies have previously been described in ewes. As far as the authors are aware, this is the first report of an ectopic pregnancy through a caesarean scar in sheep or any other farm animal species.     

Suramin alleviates hypertrophic scar in mice by inhibiting fibroplasia and inflammatory response

AIM: To investigate the therapeutical effect of suramin on hypertrophic scar (HPS) and its mechanism. METHODS: After the mouse model of HPS was established by mechanical stretching, the suramin solution at low dose (5 mg/kg) and high dose (10 mg/kg) was applied onto the scar site caused by mechanical load in mice by transdermal administration once a day for 10 d. The degree of scar hyperplasia was observed by macroscopy. The scar cross-sectional area and scar elevation index in the HPS tissues were evaluated by hematoxylin-eosin (HE) staining. The expression levels of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) in HPS tissues were detected by immumohistochemical staining. The expression level of α-smooth muscle actin (α-SMA) in HPS tissues was detected by immunofluorescence staining, RT-qPCR and Western blot. The levels of tumor necrosis factor-α (TNF-α), IL-6, IL-10 and TGF-β1 in the HPS tissues were measured by ELISA. RESULTS: Macroscopic observation showed that the surface areas of scar in the HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). HE staining results showed that the scar cross-sectional area and the scar elevation index of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.05 or P<0.01). The results of immunofluorescence staining, RT-qPCR and Western blot showed that the number of α-SMA positive cells and the mRNA and protein expression of α-SMA in scar tissues of HPS mice after treatment with suramin at low and high doses were significantly decreased (P<0.05 or P<0.01). The results of immunohistochemical staining showed that the expression levels of TGF-β1 and IL-6 in scar tissues of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). The results of ELISA showed that the levels of TNF-α, IL-6, IL-10 and TGF-β1 in the scar tissues of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). CONCLUSION: Suramin inhibits the formation of HPS, which may be related to the inhibition of fibroplasia and reduction of local inflammatory response.     

Effects of WJMSC transplantation on cesarean scar defect in a rat model

AIM: To evaluate the effects of transplantation of Wharton jelly-derived mesenchymal stem cells (WJMSCs) on SD rats with previous cesarean scar defect (PCSD). METHODS: SD rats (n=50) were randomly divided into 3 groups:10 in normal operation group (N-O group), 20 in model control group (M-C group) and 20 in model treatment group (M-T group). On the 19th day of pregnancy, thread was used for continuous suture after the caesarean section (C-S) in N-O group. After C-S, LPS (1 g/L, 1 mL/kg) was injected in uterine muscle walls and electrocoagulation (power=10 W) was used in bilateral uterine incision with interrupted suture in both M-C and M-T groups. The rats in M-T group was further divided into A and B subgroups, with 10 rats each. The rats in group A were treated by intravenous injection and vaginal perfusion, both with 0.5 mL of 1×10⁹/L WJMSCs, while the rats in group B were treated by vaginal perfusion with 1 mL of WJMSCs, once a week, totally 3 times. The rats in M-C group were also divided into A and B subgroups (10 rats per group) and treated by the same way as M-T group, only except that saline was used instead of WJMSCs. At the 1st and 4th weeks after the treatment was finished, 6 and 4 rats were executed in every group, respectively. The uterine specimens with Masson and immunohistochemical staining were observed for determining the expression of actin and cytokeratin to reveal the repair of endometrial epithelium. RESULTS: The thickness of uterine muscle and volume density of actin in M-C group were significantly lower than those in N-O group, but the muscle collagen volume density and defect rate of endometrium were higher than those in N-O group. At the 1st and 4th weeks after treatment with WJMSCs, the muscular thickness and muscular actin concentration in M-T group (A and B) were significantly higher than those in M-C group, while the number of muscular collagen fibers were significantly lower than that in M-C group. The endometrial insufficiency in M-C group was higher than that in the other groups (P<0.05). CONCLUSION: WJMSCs promote the repair of the structure and function of myometrium in PCSD rats. No obvious difference between the effects of intravenous injection combined with vaginal perfusion, and vaginal perfusion alone on the repairing is observed.     

烟台市富士苹果上苹果锈果类病毒分子变异分析

为明确苹果锈果类病毒(Apple scar skin viroid,ASSVd)在烟台市富士苹果上的变异情况,通过特异性引物对携带苹果锈果类病毒的富士嫩叶进行ASSVd全长扩增,利用生物学软件DNAMAN对所得变异序列进行分析并构建系统进化树。结果表明,从130个样品中筛选到36个阳性样品,36个阳性样品中共克隆获得52条329~333 nt的ASSVd变异序列,其中30个阳性样品含2条或2条以上的ASSVd序列。对所得变异序列进行序列比对发现,同一样品不同克隆间核苷酸序列相似性为94.0%~100.0%,所有变异序列核苷酸序列相似性为94.0%~99.7%,与Gen Bank中来自不同国家或地区的10条ASSVd分离物核苷酸序列相似性为88.3%~99.7%。将序列相似性低于97.0%的12条变异序列进行系统进化分析,结果显示除了333 nt变异序列ZS2-6与伊朗苹果分离物在同一分支外,其余11条变异序列位于同一分支。52条变异序列多序列比对发现,变异位点主要集中在TL区、P区和C区。研究表明烟台市富士苹果上ASSVd存在一定的分子变异。     

利用内标为基础的苹果锈果类病毒RT-PCR检测技术

为开发准确、灵敏的苹果锈果类病毒(Apple scar skin viroid,ASSVd)RT-PCR检测方法,以田间染病苹果组织为试材,提取高质量总RNA为模板,在常规RT-PCR检测体系中引入苹果线粒体nad5基因作为内标基因,对RT-PCR反应体系和程序进行优化,并测定其灵敏度和稳定性,最后利用优化的检测体系确定苹果果实着色期最佳检测部位。结果表明:以RNA提取改良法提取总RNA为模板合成cDNA后,进行PCR扩增,优化后的PCR体系(25μL)中cDNA模板2μL、ASSVd(20pmol/μL)正反向引物各1.0μL,nad5(20pmol/μL)正反向引物各0.1μL;扩增程序中退火温度为60.9℃,循环次数为35次;检测灵敏度为37.5ng新鲜样本;果实着色期,染病植株的幼嫩叶片、1a生枝的韧皮部和木质部、果实及部分种子样本均能检测到病毒,最佳检测部位为幼嫩韧皮部。研究结果可以为ASSVd高效、快速、准确的检测提供可靠方法,并为田间病害诊断及无毒苗木繁育提供依据和借鉴。     

基于与景观营建结合的岩仓残痕整容设计研究

指出目前的岩仓残痕治理过程通常以安全性为原则,运用工程技术,以工程材料和植被进行简单覆盖,缺乏景观艺术效果和游赏功能性。突出了岩仓残痕区整容设计的原则在于尊重自然、稳定坡地,适地适树、经济美观,特色美与协调统一美相结合,艺术性与功能性相结合,创新性与艺术性相结合,在此基础上提出整容设计的基本思路与设计方法,并以柳州市银山岩仓残痕工程治理为例,分析了当地地质及岩仓残痕现状,气候特点以及石山植物资源及植被状况,进而探讨了具体的岩仓整容设计对策。结果表明：将工程技术与景观营建结合起来,设计层次丰富、生动自然、功能完善、安全有效的园林景观,不仅易于为市民所接受,也能为其他岩仓残痕的整容设计起到示范作用。     

生物素标记cDNA探针检测苹果锈果类病毒

以含苹果锈果类病毒(ASSVd)全长序列的重组质粒PUC为模板DNA,加入ASSVd类病毒特异性引物,应用聚合酶链式反应技术(PCR)合成了生物素标记的cDNA探针,在斑点杂交反应中,探针最适使用浓度为1/10,探针检测感病的ASSVd类病毒的最大稀释倍数是1/100。利用制备的探针进行田间检测,结果与RT-PCR检测结果一致,阴性对照均无杂交信号出现。