小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag的构建及表达验证

目的构建含有生精细胞特异启动子Dazl的真核报告基因表达载体AAV-Dazl-RFP-Flag,研究生精细胞特异启动子Dazl在生精细胞中的启动效果。方法通过同源重组的方法对腺相关病毒载体AAV-CMV-RFP-Flag进行改造,构建小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag。在体外,转染GC1-spg野生型细胞和HEK-293T细胞,通过免疫荧光、q-PCR、Western blot验证AAV-Dazl-RFP-Flag在体外的表达特异性。在体内,将AAV-Dazl-RFP-Flag进行腺相关病毒(AAV)包装,通过显微注射技术将AAV注射到小鼠睾丸内,通过q-PCR检测AAV-Dazl-RFP-Flag在体内的表达特异性。结果成功构建了AAV-Dazl-RFP-Flag表达载体,并在体内、外水平验证了AAV-Dazl-RFP-Flag的表达。结论小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag可以在生精细胞中特异性表达,为雄性生殖不育的基因治疗提供更高效的载体工具。     

氧等离子体硫化法制作可变形聚二甲基硅氧烷薄膜培养基底

发展了氧等离子体硫化法制作可变形聚二甲基硅氧烷薄膜培养基底的技术。与传统的加热硫化法相比，该法制作的基底表面具有亲水性，细胞的黏附、铺展以及基底皱褶变形的发展均非常迅速。此外，观察了细胞松弛素D处理过程中基底皱褶的变化，结果表明这种技术具有较高的时间分辨率。     

Endogenous estrogen status, but not genistein supplementation, modulates 7,12-dimethylbenz[a]anthracene-induced mutation in the liver cII gene of transgenic big blue rats

A growing number of studies suggest that isoflavones found in soybeans have estrogenic activity and may safely alleviate the symptoms of menopause. One of these isoflavones, genistein, is commonly used by postmenopausal women as an alternative to hormone replacement therapy. Although sex hormones have been implicated as an important risk factor for the development of hepatocellular carcinoma, there are limited data on the potential effects of the estrogens, including phytoestrogens, on chemical mutagenesis in liver. Because of the association between mutation induction and the carcinogenesis process, we investigated whether endogenous estrogen and supplemental genistein affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in rat liver. Intact and ovariectomized female Big Blue rats were treated with 80 mg DMBA/kg body weight. Some of the rats also received a supplement of 1,000 ppm genistein. Sixteen weeks after the carcinogen treatment, the rats were sacrificed, their livers were removed, and mutant frequencies (MFs) and types of mutations were determined in the liver cII gene. DMBA significantly increased the MFs in liver for both the intact and ovariectomized rats. While there was no significant difference in MF between the ovariectomized and intact control animals, the mutation induction by DMBA in the ovariectomized groups was significantly higher than that in the intact groups. Dietary genistein did not alter these responses. Molecular analysis of the mutants showed that DMBA induced chemical-specific types of mutations in the liver cII gene. These results suggest that endogenous ovarian hormones have an inhibitory effect on liver mutagenesis by DMBA, whereas dietary genistein does not modulate spontaneous or DMBA-induced mutagenesis in either intact or ovariectomized rats.     

体外反搏治疗失血性休克中一氧化氮合酶的变化

目的：探讨反搏治疗失血休克中一氧化氮合酶（ＮＯＳ）的变化。方法：复制狗的失血休克模型，用同位素方法测定反搏前后各组织的ＮＯＳ活性。结果：体外反搏后平均动脉压较反搏前明显上升（Ｐ＜００１）。脑ＮＯＳ测定值假手术对照组明显高于失血休克组Ｐ＜００１）及失血休克克反搏组（Ｐ＜００１），失血休克组则明显低于失血休克反搏组（Ｐ＜００１）；心肌ＮＯＳ活性假手术对照组与失血休克反搏组均明显高于失血休克组（Ｐ＜００５，Ｐ＜００１），但假手术对照组与失血休克反搏组之间无显著差异（Ｐ＞００５）；主动脉ＮＯＳ活性假手术对照组明显高于失血休克组（Ｐ＜００１）及失血休克反搏组（Ｐ＜００５），但失血休克组与失血休克反搏组间无明显差异（Ｐ＞００５）。结论：体外反搏可以增强小血管ＮＯＳ活性，ＮＯＳ活性的恢复则可能在反搏治疗中起重要作用     

Possible role of a proteinase in endosporulation of Coccidioides immitis.

We previously reported isolation of a serine proteinase from the soluble conidial wall fraction of Coccidioides immitis. The purified proteinase was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, we raised monospecific antiserum in rabbits against the purified proteinase for use in immunoelectron microscopy. We showed that immunolabel was localized in the cell wall of both the saprobic and parasitic phases but was most concentrated in the wall of the segmentation apparatus of spherules just prior to endospore differentiation. The total wall fractions of the mycelial phase, as well as those of presegmented and endosporulating spherules, were isolated from in vitro grown cells and then treated with a proteinase inhibitor (phenylmethylsulfonyl fluoride [PMSF]) which irreversibly binds to the residual proteolytic enzyme in the wall isolates. Each fraction was dialyzed, lyophilized, and separately incubated with the active, purified 36,000-Mr proteinase. The reaction mixtures were examined spectrophotometrically (A280) for decomposition of the substrates. Only the PMSF-treated wall isolated from endosporulating spherules was significantly digested. Active, 36,000-Mr proteinase was isolated from intact and viable, endosporulating spherules by brief extraction of the cells with 1% octyl-beta-D-thioglucoside, a nonionic detergent. The serine proteinase may be partly responsible for autolysis of the segmentation apparatus of mature spherules, a morphogenetic process which is pivotal for release of endospores and subsequent proliferation of the pathogen.     

聚焦超声对卵巢癌细胞株COC1细胞凋亡和Bcl-2/bax基因表达的影响

目的:研究低频脉冲的聚焦超声对人卵巢癌细胞株COC1的抗肿瘤作用,并探讨其诱导细胞凋亡的分子机制.方法:以低频脉冲聚焦超声(超声频率0.8 MHz,声强83.6 W/cm2,脉冲持续时间50μs)辐照卵巢癌COC1细胞10秒,采用MTT法观察超声对COC1细胞增殖的抑制作用;流式细胞仪(FCM)、TUNEL法、荧光染色和光、电镜检测细胞凋亡;应用FCM免疫荧光技术观察Bcl-2、bax基因蛋白表达的变化.结果:低频脉冲聚焦超声可明显抑制存活卵巢癌COC1细胞株的增殖,辐照后12 h、24 h、36 h和48 h,COC1细胞MTT光吸收值与对照组相比显著降低;FCM检测超声辐照后6 h的COC1细胞凋亡率达(23.57±4.62)%;Bcl-2蛋白的荧光指数(FI)降低,而bax蛋白FI升高.同时TUNEL法观察到棕褐色着染的凋亡细胞;荧光显微镜和电镜下观察到凋亡小体形成等典型的形态学改变.结论:低频脉冲聚焦超声辐照对人卵巢癌细胞株COC1具有细胞毒作用,其机制为诱导辐照后存活的人卵巢癌细胞株COC1凋亡,bcl-2和bax蛋白可能参与了COC1细胞凋亡过程.     

不同机制大动脉粥样硬化型脑卒中基线特征和血管内治疗后结局比较

目的 本研究旨在比较颅内大血管伴/不伴同侧颈内动脉闭塞的大动脉粥样硬化型脑卒中患者基线特征以及行血管内治疗后结局的差异。方法 对DIRECT-MT亚组进行回顾性分析,以比较前循环大动脉粥样硬化（large-artery atherosclerosis,LAA）型卒中串联闭塞和颅内闭塞接受血管内治疗（endovascular treatment,EVT）的患者的基线特征和预后,分析不同机制学特征（动脉粥样硬化或动脉-动脉栓塞）对临床结局的影响。结果 LAA型卒中患者共108例,其中串联闭塞63例,颅内闭塞45例。颅内闭塞组患者高血压史率高于串联闭塞组（77.8% vs. 52.4%, P=0.007）。颅内闭塞组闭塞部位最常见于大脑中动脉M1段（88.6%）,而串联闭塞组颅内闭塞主要位于颈内动脉颅内段（49.2%）和大脑中动脉M1段（49.2%）（P＜0.001）。两组患者在年龄、性别、术前抗栓、他汀类药物的使用,卒中、房颤、吸烟史,基线mRS、NIHSS评分,是否静脉溶栓,侧枝循环,以及救治流程时间差异均无统计学意义（P均＞0.05）。90天mRS 0-2分的患者比例两组差异无统计学意义（53.3% vs. 41.9%, P=0.243）。颅内闭塞组术后成功再灌注率高于串联闭塞组（93.3% vs. 77.4%, P=0.026）,但术后24-72小时血管再通的比例前组低于后组（57.1% vs. 77.2%, P=0.034）。最终梗死体积,颅内闭塞组小于串联闭塞组（20.1 vs. 34.5, P=0.025）。术后NIHSS评分,90天EQ-5D-5L评分和BI指数等其他次要结局,两组间差异无统计学意义（P均＞0.05）。两组在90天内的死亡率,发生的无症状性和症状性颅内出血率,5-7天时在另外的血管区域新发脑梗死,以及新流域栓塞的患者百分比相似,差异无统计学意义（P均＞0.05）。结论 动脉粥样硬化导致的串联闭塞相较于孤立颅内闭塞,末次造影成功再灌注率较低,梗死体积更大,但术后24-72小时再通率更高,且神经功能良好预后率以及不良事件发生率均与颅内闭塞相仿。     

Homer 1b/c通过内质网功能调节由谷氨酸诱发的HT22细胞自噬

目的　研究Homer1b/c蛋白在谷氨酸诱发的细胞自噬中的作用及机制。方法　选用小鼠海马细胞系HT22细胞,通过500 ?mol/L谷氨酸处理建立细胞损伤模型。用siRNA慢病毒转染方式下调Homer1b/c表达和10 ?mol/LBAPTA-AM（1,2-双(2-氨基苯氧基)乙烷-N,N,N`,N`-四乙酸四乙酸甲酯,钙离子螯合剂）、10 mmol/L4-PBA（4-苯基丁酸,内质网应激抑制剂）分别抑制细胞内钙离子释放和内质网应激后,使用蛋白质印迹法检测Homer1b/c,自噬蛋白Beclin-1、微管相关蛋白轻链3（LC3）,以及内质网应激标志蛋白CHOP（人内质网应激相关蛋白）、GRP-78(葡萄糖调节蛋白78)的表达水平。每组实验均进行3次,采用独立样本t检验和单因素方差分析进行统计学分析。结果　谷氨酸处理HT22细胞12 h后,Beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值均升高（P<0.05）,下调Homer1b/c表达可降低Beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值的升高程度（P<0.05）。抑制细胞内钙离子释放和抑制内质网应激均能降低Beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值的升高程度（P<0.05）。然而在下调Homer1b/c表达后,抑制细胞内钙离子释放和抑制内质网应激未能进一步降低Beclin-1表达和LC3-Ⅱ/LC3-Ⅰ比值的升高程度（P<0.05）。结论　Homer1b/c能够调节谷氨酸诱导的自噬,其调节作用可能与内质网功能有关。     

3D打印导板辅助上颈椎椎弓根置钉与徒手置钉的比较

目的 比较个体化3D打印导板辅助上颈椎椎弓根置钉与徒手置钉的精准性。方法 选择2019年6月至2022年6月在蚌埠医学院第二附属医院就诊的上颈椎疾患患者20例,获取每位患者颈椎三维CT数据,根据置钉方式不同分为导板组和对照组,分别制作20例等比例3D打印上颈椎模型,导板组使用3D打印导板辅助上颈椎模型椎弓根螺钉置入,对照组在透视辅助下徒手置钉。根据椎体是否存在骨折,将导板组内20例模型分为钉道损伤组10例（寰椎骨折与枢椎骨折病例）和钉道完整组10例（畸形与关节脱位病例）。比较导板组和对照组螺钉可接受率、单枚螺钉置入时间差异;比较导板组术前理想钉道与术后实际钉道内倾角差异,对比钉道损伤组和钉道完整组置钉结果差异。结果 导板组螺钉可接受率为93.55%,高于对照组的79.03%(P<0.05);导板组单枚螺钉置入时间短于对照组（P<0.05）;导板组术前理想螺钉内倾角与术后实际螺钉内倾角之间差异无统计学意义（P>0.05）;钉道损伤组螺钉突破椎弓根率26.7%高于钉道完整组3.1%(P<0.05)。结论 个体化3D打印导板辅助上颈椎椎弓根置钉在置钉精准度、安全性和置...     

基于倾向性匹配法的胆囊结石合并慢性胆囊炎手术DRG控费效果研究

目的 基于倾向性匹配分析疾病诊断相关分组（DRG）对胆囊结石合并慢性胆囊炎手术患者住院费用的控费效果。方法 收集2021年1月至2022年12月安徽医科大学第一附属医院1 010例胆囊结石合并慢性胆囊炎手术患者住院费用数据,按付费方式分为DRG组（n=570）和非DRG组（n=440）。采用描述性统计分析、倾向性评分法、秩和检验,平衡病例间混杂因素后,检验DRG组和非DRG组住院费用情况。结果 共纳入1 010例病例,经倾向性匹配成功852例。胆囊结石合并慢性胆囊炎患者DRG组与非DRG组年龄、住院天数在匹配前差异有统计学意义（P<0.05）。患者住院费用中,耗材费、药费占比较高。经倾向性匹配后,DRG组与非DRG组住院总费用（Z=7.519,P<0.001）、自费金额（Z=13.903,P<0.001）、医保报销费用（Z=11.561,P<0.001）、综合医疗服务费（Z=2.188,P=0.029）、手术费（Z=2.256,P=0.024）、药费（Z=3.202,P=0.001）、耗材费（Z=2.229,P=0.026）、诊断费（Z=8.263,P<0.001）差异均有统计学意义。结论 DRG对胆囊结石合并慢性胆囊炎患者自费金额、综合医疗服务费、手术费、药费、耗材费及诊断费均具有控费效果,且总体对住院费用控费效果较好,但费用结构方面仍存在优化空间。