Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.
Gene Targeting ; Green Fluorescent Proteins ; genetics ; MicroRNAs ; genetics ; Oryza ; genetics ; Plasmids ; Protoplasts ; metabolism ; RNA, Plant ; genetics ; Transfection

Objective To investigate the effect of intrathecal glial cell line-derived neurotrophic factor (GDNF) on p38 mitogen-activated protein kinase (p38MAPK) protein expression in the spinal cord dorsal horn in a rat model of neuropathic pain. Methods Male SD rats 6 weeks old weighing 180-200 g were used in this study.One hundred and twenty rats in which intrathecal catheters was successfully implanted were randomly divided into 4 groups ( n = 30 each): group Ⅰ control ( group C); group Ⅱ sham operation ( group S); group Ⅲ neuropathicpain (group P) and group Ⅳ GDNF. In groupⅢ and Ⅳ I6 spinal nerve was ligated. In group Ⅳ intrathecal GDNF was administered every other day for 14 d after spinal nerve ligation. In group Ⅲ normal saline was given instead. Ten animals from each group were selected at 3, 7 and 14 d after spinal nerve ligation, the mechanical pain threshold was measured, and then the rats were decapitated. The I4-6 segment of the spinal cord of the operated side was isolated. p38MAPK protein expression in the spinul dorsal horn was determined by immunohistochemistry and Western blotting. Results Intrathecal GDNF significantly attenuated spinal nerve ligationinduced increase in p38MAPK protein expression in spinal dorsal horn of the operated side. Conclusion IT GDNF can relieve neuropathic pain by inhibiting p38MAPK protein expression in the spinal dorsal horn.

Objective To study the application effect of feedback training mode on new neonatal recovery technology.Methods 101 midwives,obstetric doctors and pediatricians who participated the training were tested about the professional knowledge and skills of new neonatal recovery technology according to the teaching material of neonatal asphyxia recovery and neonatal resuscitation guidelines (2007 revision in Beijing).The results showed no statistical significance.And they were randomly divided into the control group (51 cases) and the observation group(50 cases).The control group adopted traditional method,that was,the teacher taught first and then the students took practice.The observation group used feedback training method.Results The theoretical and technical results of the observation group were significantly higher than those of the control group.The learning motivation,learning initiative,cooperation degree,ability of observation and solving problem and operation proficiency were also significantly higher than those of the control group.Conclusions Feedback training methods can significantly increase the learning effect of perinatal personnel,and make popularized the new neonatal recovery technology.

Objective　To study the value of B type ultrasonography guiding percutaneous radiofrequency ablation (PRFA) for hepatic cancer (HC). Methods　70 patients with primary or metastatic hepatic malignancies were treated by PRFA under ultrasound guidance through LeVeen multipolar array needle electrode and RF 2000. All patients were followed up by turnor marker, B type sounography and MRI monthly. Results　PRFA were performed in 53 patients with hepatocellular carcinoma (HCC) and 17 cases with metastatic liver cancer. Liver function classfication was 53 cases in Child class A, 15 in class B and 2 in class C. In 21 patients with non-operated small HCC (≤5 cm), 12 of 17 patients with AFP positive were decreased to normal (70.6%) and decreased markedly in 4 (23.5%), and MRI showed the treated tumors were completely necrosis in 16 patients (76.2%). The 3, 6 month survival rates were 90.5%, 100% respectively in small HC groups, and 66.7% to 72.7% and 27.6% to 72.7% in large HC groups.Conclusions　PRFA as a local thermal therapy is becoming a kind of new palliative treatment for hepatic malignancies, which not only is safe, minimally invasive, and effective for small ones; but also can be used with combination of TACE for larger ones.

Objective To explore the best time period of pulse oximetry in congenital heart disease (CHD) screening. Methods Totally 5433 newborns delivered or treated in Ningbo Women and Children’s hospital were enrolled in the study. Cardiac color ultrasound was used to arrive diagnosis and every screening time period (0?~?24 h、24 h?~?48 h、48 h?~?72 h) was analysed. Results Among the three time periods: the sensibility was in the range of 54.72%?~?67.92%, specificity was 99.11%?~?99.61%, false negative rate was 32.08%?~?43.40%, false positive rate was 0.39%?~?0.89%, Youden index was 0.54?~?0.68, coincidence rate was 98.67?~?99.30, and kappa was 0.44?~?0.65. Conclusion The best screening time is the period between 48h and 72 h after birth.

Objective To explore clinical application of retrograde medial and lateral gastrocnemius muscle flap for soft tissue defects of the middle and lower third of the leg. Methods From August 2008 to December 2009, in our hospital we adopted retrograde medial and lateral gastrocnemius muscle flap to renovate 5 cases of refractory soft tissue defects of the middle and lower third of the leg. Results Five cases of retrograde medial gastrocnemius muscle flap were survived, morphology and function of soft tissue defects were renovated well. Conclusion This operation is an effective and reliable technique for soft tissue defects of the middle and lower third of the leg, which is performed without sacrificing the major blood vessels, probing vascular pedicle and matching vascular anastomosis.

Objective To investigate the effects of macrophage migration inhibitory factor (MIF) overexpression on the expression of interleukin-8 (IL-8),martix metalloproteinase-9 (MMP-9) and invasion of human cervical cancer SiHa cells.Methods Chemical synthesis MIF eDNA gene,designed primer sequence including XhoI and BamHI enzyme sites,MIF gene was amplified by polymerase chain reaction (PCR),constructed eukaryotic expression vector pEGFP-N1/MIF and transfected into SiHa cells using Lipofectamine and won over-expression of MIF.The expression of MIF in supernatant fluid was detected by ELISA,the expression of MIF,IL-8,MMP-9 in both mRNA and protein levels were detected by real-time fluorescence quantitative-PCR and immunocytochemistry respectively.The effect of over-expressed MIF on migration was detected by Boyden small chamber.Results The expression of protein in supernatant fluid transfected with pEGFP-N1/MIF was significantly increased (Fgroup =8267.564,P ＜ 0.01),the expression of MIF,IL-8,MMP-9 in both mRNA and protein in SiHa cells transfected with pEGFP-N1/MIF were significantly increased (F values were 7019.619,2148.094,3303.540,1565.114,2807.300,523.466,P ＜ 0.01),and there was a positive correlation among MIF,IL-8,MMP-9 expression in both mRNA and protein (r values were 0.865,0.895,0.934,0.908,P ＜ 0.01).Invasion ability in SiHa cells transfected with pEGFP-N1/MIF was obviously increased (F=3430.898,P＜ 0.01).Conclusion The over-expression MIF gene in SiHa cells can promote cervical cancer cell invasion and metastasis of ability,which could be associated with the upregulation of IL-8 and MMP-9 expression.

Background Primary open angle glaucoma (POAG) is a major blindness-causing disease,characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork cells has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye (POAG) and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunochemistry with the laminin (LM), fibronectin (FN) and neuron specific endolase (NSE)monoclonal antibodies. The ultrastructure was examined to observe the biological characteristics of the cells under the transmission electronic microscope. The experimental results were compared among POAG group, normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were 0. 35 ± 0.06 and 0. 26 ± 0. 01, and those for LM were 0. 34 ± 0. 03 and 0. 25 ± 0. 02 respectively, showing statistically significant difference between these two groups ( FN: t = 14. 446, P＜0.001; LM: t = 9. 346, P＜0. 001 ). The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork cells from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.

Objective:To study the effects of Docetaxel o n the proliferation of adenoid cystic carcinoma SACC-83 cells of salivary gland. Methods:The inhibitory effects of Docetaxel on the proliferatio n of SACC-83 cells were investigated with cell counting, soft agar clonogenic a ssay, and flow cytometry. Results:With the exposure time of 24, 48 or 72 h the IC 30(nmol/L) of Docetaxel was 1.39,1.26 and 0.47, the I C 50(nmol/L) 13.02, 3.34 and 1.26 respectively; the relative antitumor acti vity (RAA) of the drug against SACC-83 cells was 330, 1 289 and 3 426 respectiv ely. After the cells had been treated for 72 h, the percentages of G 1, S and G 2 phase cells in the cell cycle in the control group were 73.8,19.8 and 6.4, in IC 30 group 65.0, 29.5 and 5.5, in IC 50 group 57.6,42.4 and 0, res pectively. The clonogenesity (%) in control, IC 30 and IC 50 groups we re 36.0?0.5,8.3?2.5 and 0.5?0.3 respectively. Conclusion:Doc etaxel may inhibit the proliferation of SACC-83 cells in a dose and time depend ent way.

Sleep disorders are commonly occurred among patients with Parkinson's disease,such as difficulties in the initiation of sleep,fragmented sleep,sleep behavior disorder and excessive daytime sleepiness.The mechanism of sleep disorders associated with Parkinson's disease is not clear,which may be associated with the injury of brain stem,nuclei of median raphe,nuclei fasciculi solitarii,thalamencephalon and the changes of neurotransmitters as dopamine,hypocretin(orexin) and melatonin.This article gives an overview of the mechanism of sleep disorders associated with Parkinson's disease.