靶向EZH2基因的shRNA真核表达载体的构建及鉴定

目的构建靶向EZH2基因的shRNA真核表达载体质粒,为利用RNA干扰技术探索EZH2基因的作用的研究做准备。方法根据EZH2 mRNA序列设计并合成shRNA寡核苷酸片段,退火形成双链并连接入pGenesil-2载体,并进行酶切鉴定和测序。结果酶切证明构建的shRNA已插入载体中,经测序证明与设计相同。结论成功构建靶向EZH2基因的shRNA真核表达载体,为进一步研究EZH2基因在肿瘤细胞中的作用机制及后续的体内外RNAi实验研究奠定了基础。     

TGF-β和ERK在骨质疏松大鼠骨组织中的表达

目的:研究转化生长因子(TGF-β)和信号调节激酶(ERK)在去卵巢大鼠骨组织中的表达及意义。方法:30只2月龄SD雌性大鼠分别造模为假手术组(SHAM组)、卵巢切除组(OVX组)及雌激素替代组(OVX-N组)各10只,用免疫组化法测定成骨细胞中TGF-β和磷酸化ERK的表达并进行定量分析。结果:TGF-β蛋白阳性表达主要在成骨细胞的胞膜上和胞浆内,ERK主要在成骨细胞核内,OVX-N组TGF-β和ERK的阳性表达高于OVX组(P<0.01,0.05),与SHAM组比较差异不明显。结论:TGF-β和ERK可能在绝经后骨质疏松症中起重要调节作用。     

Suppression of WNK1-SPAK/OSR1 Attenuates Bone Cancer Pain by Regulating NKCC1 and KCC2

Our preliminary experiment indicated the activation of with-nolysine kinases 1 (WNK1) in bone cancer pain (BCP) rats. This study aimed to investigate the underlying mechanisms via which WNK1 contributed to BCP. A rat model of BCP was induced by Walker-256 tumor cell implantation. WNK1 expression and distribution in the lumbar spinal cord dorsal horn and dorsal root ganglion were examined. SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress-responsive kinase 1 (OSR1), sodium-potassium-chloride cotransporter 1 (NKCC1), and potassium-chloride cotransporter 2 (KCC2) expression were assessed. Pain behaviors including mechanical allodynia and movement-evoked pain were measured. BCP rats exhibited significant mechanical allodynia, with increased WNK1 expression in the dorsal horn and dorsal root ganglion neurons, elevated SPAK/OSR1 and NKCC1 expression in the dorsal root ganglion, and decreased KCC2 expression in the dorsal horn. WNK1 knock-down by small interfering alleviated mechanical allodynia and movement-evoked pain, inhibited WNK1-SPAK/OSR1-NKCC1 activities, and restored KCC2 expression. In addition, closantel (a WNK1-SPAK/OSR1 inhibitor) improved pain behaviors, downregulated SPAK/OSR1 and NKCC1 expression, and upregulated KCC2 expression in BCP rats. Activation of WNK1-SPAK/OSR1 signaling contributed to BCP in rats by modulating NKCC1 and KCC2 expression. Therefore, suppression of WNK1-SPAK/OSR1 may serve as a potential target for BCP therapy.PerspectiveOur findings demonstrated that the WNK1-SPAK/OSR1 signaling contributed to BCP in rats via regulating NKCC1 and KCC2. Suppressing this pathway reduced pain behaviors. Based on these findings, the WNK1-SPAK/OSR1 signaling may be a potential target for BCP therapy.     

Formation of Methylated and Phosphorylated Metabolites During the Fermentation Process of Verdamicin

In an attempt to understand the biosynthetic processes leading to the formation of verdamicin (end product), we have examined the patterns of the formation of methylated and phosphorylated metabolites, which resulted from either the addition of l-[methyl-¹⁴C]methionine or [³²P]KH₂PO₄ to the fermentation. Incorporation of label from l-[methyl-¹⁴C]methionine into the bioactive sisomicin, verdamicin, and the chromatographically polar components increased with the progression of time. Two methylated bioinactive metabolites were found in the culture broth after removal of the methylated bioactive metabolites. In contrast to the bioactive metabolites, incorporation of the methyl-¹⁴C label into the two methylated bioinactive metabolites decreased with the progression of time. A phosphorylated bioinactive metabolite (nonmethylated) was also found in the culture broth, fermented in the presence of [³²P]KH₂PO₄. The role of the phosphorylated metabolite in the biosynthesis of the bioactive metabolites cannot yet be explained.     

Evaluation of New Bone Formation in Normal and Osteoporotic Rats with a 3-mm Femur Defect: Functional Assessment with Dynamic PET-CT (dPET-CT) Using 2-Deoxy-2-[18F]Fluoro-d-glucose (18F-FDG) and 18F-Fluoride

Purpose

The aim of the current study was to assess the formation of new bone in a 3-mm created defect in the femur and its adjacent bone tissue in osteoporotic and normal animals. The assessment is based on bone remodeling and glucose metabolism in a rat model with a 3-mm created defct in the femur using ¹⁸F-fluoride and 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG) as tracers for dynamic PET-CT (dPET-CT). The ¹⁸F-fluoride PET data were compared with those of ¹⁸F-FDG.

Procedures

Osteoporosis was induced by ovariectomy and a calcium restricted diet in each rat (n?=?7). Alternatively, a sham operation was performed in the control group (n?=?8). After 3 months, all rats were operated to create a 3-mm defect using an oscillating saw in the distal metaphyseal femur, which was internally fixed with a metal plate. Eighteen weeks after osteoporosis induction and 6 weeks following femoral surgery, dPET-CT studies scan were performed with ¹⁸F-FDG and ¹⁸F-fluoride. Following PET data acquisition, standardized uptake values (SUVs) were calculated from the tracer concentration values. Then, a two-tissue compartmental learning-machine model was applied to the data for the calculation of the compartment parameters (K1-k4, VB, Ki). Furthermore, a non-compartmental model based on the fractal dimension was applied for quantitative analysis of both groups and both tracers. Finally, multivariate analysis was performed for the statistical analysis of the kinetic data.

Results

The values for K1 and Ki were higher in the osteoporotic rats than in the control group. Ki and K1 of ¹⁸F-fluoride in the adjacent bone tissue differ significantly based on the Wilcoxon rank-sum test for the osteoporotic and control group (p?<?0.05). The sensitivity and the negative predictive value (NPV) based on linear discriminant analysis was high with a value of 100 % for both tracers and both evaluated regions (defect and adjacent bone tissue) when comparing control and osteoporotic rats. The overall accuracy with ¹⁸F-FDG was generally higher than that with ¹⁸F-fluoride for both evaluated regions for the control and osteoporotic rats based on a multiparameter evaluation.

Conclusion

In this study, the changes in tracer kinetics accurately discriminated differences in the created defect in the femur and its adjacent bone tissue between osteoporotic and control rats.     

正骨手法结合MIPPO技术重建内侧柱稳定治疗老年骨质疏松性肱骨近端骨折的对照研究

目的:探讨正骨手法闭合复位结合经皮微创固定(MIPPO)治疗老年肱骨近端骨折的临床疗效。方法:选取老年肱骨近端骨折40例随机分为正骨手法+MIPPO技术固定组(简称手法组)和传统切复内固定组(简称切复组),每组20例,分别进行治疗。术后3、6、12个月,观察恢复情况:骨折恢复、内固定松动断裂、肱骨头缺血坏死、内固定取出时间和内固定取出后局部骨质等情况,及有无再骨折,并采用Constant-Murley评分系统对肩关节功能进行评分。结果:40例患者均获随访,手法组1例患者术后出现肱骨头内翻畸形,骨折畸形愈合,1例患者出现肱骨头缺血性坏死;切复组2例患者出现肱骨头内翻。两组术后肩关节功能Constant-Murley评分比较,差异有统计学意义(P0.05)。结论:正骨手法闭合复位结合经皮微创固定治疗老年肱骨近端骨折,重建内侧柱稳定,具有创伤小、血循破坏少、固定可靠等优点,是治疗老年性肱骨近端骨折的有效方法。     

bcl—2反义寡核苷酸增强K562白血病细胞对As2O3药物敏感性

采用细胞培养法,免疫组化实验技术和流式细胞仪等项技术,研究了bcl-2基因反义核酸和砷剂单独及联合应用对K562细胞的生物效应,DNA及bcl-2蛋白的变化.结果显示,As2O3(2.0μmol/L)+bcl-2 ASODN(10.0μmol/L)联合作用比单用As2O3(2.0 μmo1/L)或bcl-2 ASODN(10.0μmol/L)显著增强诱导细胞凋亡的作用(P＜0.01);流式细胞仪检测显示,联合作用引起的bcl-2蛋白表达亦明显减少(P＜0.1).结论表明,bcl-2基因反义核酸能明显提高和显著增强K562白血病细胞对As2O3药物敏感性.     

Analysis of Apparent Integrated Backscatter Coefficient and Backscattered Spectral Centroid Shift in Calcaneus in vivo for the Ultrasonic Evaluation of Osteoporosis

The purposes of our study were to evaluate the correlation among apparent integrated backscatter coefficient (AIB), spectral centroid shift (SCS) of ultrasonic backscatter signals and bone mineral density (BMD) and to examine the effectiveness of ultrasound variables as predictors of osteoporosis. A total of 1011 persons aged 21–80 y old were included. All study participants underwent BMD measurements of the lumbar spine (LSBMD) and the femoral neck (FNBMD). The participants also underwent calcaneal measurements to determine AIB and SCS with central frequencies of 3.5 (one transducer) and 5.0 MHz (the other transducer). AIB decreased with age and was positively correlated with BMD, while SCS increased with age and was negatively correlated with BMD. The correlation coefficient of SCS with LSBMD and FNBMD at 3.5 MHz was −0.72 and −0.70, respectively. The correlation coefficient at 5.0 MHz was −0.75 and −0.74, respectively. The correlation coefficient of AIB with LSBMD and FNBMD at 3.5 MHz was 0.65 and 0.63. The correlation coefficient at 5.0 MHz was 0.59 and 0.55, respectively. The correlation between SCS and BMD was significantly better than the correlation between AIB and BMD. Using receiver operating characteristic analysis, a significant difference was found between the areas under the curve for SCS and AIB at 3.5 MHz (0.781 vs. 0.715, respectively, p < 0.05), as well as at 5.0 MHz (0.782 vs. 0.709, respectively, p < 0.05). The optimum T-score threshold for SCS was -1.3 for both transducers. The sensitivity and specificity of SCS at 3.5 MHz and 5.0 MHz for the optimum threshold were 64%, 85%, 63% and 86%, respectively. In conclusion, the correlations among the ultrasound parameters and BMDs are strong. SCS performs better than AIB in differentiating patients with osteoporosis. Ultrasound variables may be taken into consideration as predictors of osteoporosis in the future considering its portability.     

沉默血管内皮生长因子及促血管生成素-2基因抑制裸鼠人肺腺癌移植瘤生长的实验研究

目的研究沉默血管内皮生长因子(VEGF)及促血管生成素-2(Ang-2)基因表达对裸鼠人肺腺癌移植瘤生物学特性的影响。方法利用基因重组技术构建H1启动子驱动表达针对VEGF及Ang-2siRNA的重组腺病毒Ad-VEGFshRNA及Ad-Ang-2shRNA。制备裸鼠人肺腺癌A549细胞移植瘤模型,观察RNA干扰后(RNAi)对肿瘤生物学特性的影响。结果重组腺病毒接种裸鼠30d后,Ad-VEGFshRNA、Ad-Ang-2shRNA干扰组与对照组比较,肿瘤体积及重量均明显减小,差异均有统计学意义(P<0.01),并且Ad-VEGFshRNA及Ad-Ang-2shRNA联合干扰组与Ad-VEGFshRNA、Ad-Ang-2shRNA干扰组比较能够更有效地抑制肿瘤细胞的生长,差异有统计学意义(P<0.05);而Ad-VEGFshRNA组与Ad-Ang-2shRNA组比较差异无统计学意义(P>0.05)。免疫组织化学染色显示:Ad-VEGFshRNA、Ad-Ang-2shRNA干扰组细胞增殖减慢,凋亡增加,微血管密度明显少于对照组,差异有统计学意义(P<0.01)。并且Ad-VEGF shRNA及Ad-Ang-2shRNA联合干扰组与单基因干扰比较,能够更有效地抑制肿瘤细胞增殖,促进凋亡,差异有统计学意义(P<0.05)。结论 VEGF及Ang-2基因靶向RNA干扰在体内有明显抑制肺腺癌细胞生长的作用,联合抑制VEGF及Ang-2基因的表达能够更有效地抑制肺腺癌的生长。     

hsa_circ_0006950 调控miR-124-3p/EZH2 轴促进胰腺癌细胞增殖与迁移的机制研究

目的　检测环状核糖核酸（circular RNA ,cicrRNA）hsa_circ_0006950 在胰腺癌（pancreatic cancer,PC）中的表达,探讨其对胰腺癌细胞增殖、迁移的影响及其潜在分子作用机制。方法　利用实时荧光定量PCR(qRT-PCR) 检测hsa_circ_0006950 在胰腺癌组织及细胞中的相对表达水平;转染hsa_circ_0006950 干扰载体构建低表达细胞系,通过CCK-8实验、克隆斑点形成实验和Transwell 实验检测hsa_circ_0006950 对胰腺癌细胞增殖、克隆形成及迁移的影响;利用生物信息学网站预测hsa_circ_0006950 的miRNA 分子靶标及其调控网络（hsa_circ_0006950-miR-124-3p-EZH2）,双荧光素酶基因报告实验验证hsa_circ_0006950 和miR-124-3p,miR-124-3p 和EZH2 的靶向结合关系;转染过表达/ 干扰miR-124-3p 和过表达EZH2 细胞系,探究miR-124-3p 和EZH2 及hsa_circ_0006950 调控miR-124-3p/EZH2 轴对胰腺癌细胞克隆形成及迁移的影响。结果　胰腺癌组织中hsa_circ_0006950 表达明显高于癌旁正常组织（3.57±0.52 vs 1.01±0.03）,差异有统计学意义（t=21.980,P ＜ 0.001）。胰腺癌细胞PANC-1（7.51±0.62）,AsPC-1（5.26±0.45）,Capan-2（3.69±0.38）,SW1990（3.25±0.32）and BXPC-3（3.86±0.35）中hsa_circ_0006950 相对表达明显高于正常胰腺导管上皮细胞HPDE6-C7（1.00±0.01）中的表达,差异有统计学意义（F=88.585,P ＜ 0.001）。与对照组相比,干扰hsa_circ_0006950 表达组细胞增殖能力（0.79±0.17 vs 1.83±0.42）,克隆形成率（51.42%±5.84% vs 78.76%±13.65%）和迁移数目（104.64±24.73 vs 218.21±31.57）明显降低,差异均有统计学意义（t=3.976,3.190,4.905,P=0.016,0.033,0.008）。miR-124-3p 是hsa_circ_0006950 的下游靶基因,EZH2 是miR-124-3p 的直接靶标。hsa_circ_0006950 靶向负调控miR-124-3p,miR-124-3p 靶向负调控EZH2。与对照组相比,过表达miR-124-3p 组PC 细胞增殖（0.21±0.16 vs1.75±0.47）,克隆形成率（47.85%±4.13% vs 81.54%±2.33%）和细胞迁移数目（118.74±24.65 vs 202.36±31.45）明显降低,差异均有统计学意义（t=5.378, 14.317, 3.390, 均P ＜ 0.001）;共转染过表达EZH2 后,miR-124-3p 对细胞增殖、克隆形成率及迁移能力的抑制作用被逆转。在干扰hsa_circ_0006950 组细胞中同时下调miR-124-3p 或过表达EZH2 后,hsa_circ_0006950 对细胞克隆形成及迁移的抑制作用被逆转恢复。结论　胰腺癌中hsa_circ_0006950 显著高表达,抑制其表达可以抑制胰腺癌细胞的增殖及迁移;hsa_circ_0006950 可能通过靶向下调miR-124-3p 表达,进而上调EZH2 表达来发挥作用,参与胰腺癌的发生发展。     

五班交接本与护理记录单在护理交接班中的应用

《病历书写基本规范(试行)》的出台,对护理文件的书写提出了新的要求,而传统的交班本已不能达到规范的护理文书书写要求,它仅限于记录病情,白天、前夜班、后夜班各记录一次,实际工作中很多问题就会被忽视,影响护理质量。为此,笔者从2004年6月至2005年4月在大内科片区试行了五班交接本与护理记录单相结合的交班方式,收到较好的效果。