心内直视术中鱼精蛋白毒性反应的临床研究

目的：回顾性分析心内直视术鱼精蛋白毒性反应发生情况，以总结临床特点，找出防治对策。方法：近两年连续1105例心内直视手术中，发生鱼精蛋白毒性反应53例，将其分为有反应与无反应组，轻度与中重反应组，并比较其临床特点，结果：鱼精蛋白毒性反应发生率为4．8％，有反应有无反应组，轻度与中重度反应组临床一般情况无明显差别，仅中重度反应组的主动脉阻断时间与鱼精蛋白毒性反应持续时间明显较轻度反应组长（P＜0．05），中重度反应组死亡率达50％，结论：心内直视术中鱼精蛋白毒性反应发生率高，应引起临床上足够的重视，鱼精蛋白毒性反应的发生难以预测，中重度反应死亡率高，预后差，鱼精蛋白用量应适度，充分认识鱼精蛋白毒性反应的临床特点和及时再转机车不有助于防治此类事件发生。     

超低微浓度菲立磁-硫酸鱼精蛋白复合物标记骨髓间充质干细胞的方法

目的 探索简单、无细胞外铁产生的超低微浓度菲立磁-硫酸鱼精蛋白标记骨髓间充质干细胞(MSCs)方法。 方法 贴壁法培养大鼠MSCs。待3代细胞汇合至80%~90%时,更换无血清培养液,根据菲立磁和硫酸鱼精蛋白的不同浓度分为4组:A组[(7.50∶1.00)μg/ml]、B组[(10.00∶1.20)μg/ml];C组[(15.00∶1.80)μl/ml]和空白对照组,加入培养液,混匀,5% CO₂孵育15 min,补加血清后孵育至次日。检测细胞标记率、细胞内外铁、细胞活力和标记细胞MR信号。 结果 B组可有效标记大鼠MSCs,普鲁士蓝染色阳性率100%,无细胞外铁产生,铁颗粒分布于溶酶体内。4组间台盼蓝拒染率差异无统计学意义(P>0.05);体外MR GRE T2^*WI序列可检测到1×10⁴个标记细胞。 结论 使用超低微浓度菲立磁10.00 μg/ml与鱼精蛋白1.20 μg/ml可有效标记大鼠MSCs,体外MR可检测到1×10⁴个标记细胞。     

Review: Properties of sperm and seminal fluid,informed by research on reproduction and contraception

Forensic DNA testing is grounded in molecular biology and population genetics. The technologies that were the basis of restriction length polymorphism testing (RFLP) have given way to PCR based technologies. While PCR has been the pillar of short tandem repeat (STR) methods and will continue to be used as DNA sequencing and analysis of single nucleotide polymorphisms (SNPs) are introduced into human identification, the molecular biology techniques in use today represent significant advances since the introduction of STR testing. Large forensic laboratories with dedicated research teams and forensic laboratories which are part of academic institutions have the resources to keep track of advances which can then be considered for further research or incorporated into current testing methods. However, many laboratories have limited ability to keep up with research advances outside of the immediate area of forensic science and may not have access to a large university library systems. This review focuses on filling this gap with respect to areas of research that intersect with selected methods used in forensic biology.The review summarizes information collected from several areas of the scientific literature where advances in molecular biology have produced information relevant to DNA analysis of sexual assault evidence and methods used in presumptive and confirmatory identification of semen. Older information from the literature is also included where this information may not be commonly known and is relevant to current methods. The topics selected highlight (1) information from applications of proteomics to sperm biology and human reproduction, (2) seminal fluid proteins and prostate cancer diagnostics, (3) developmental biology of sperm from the fertility literature and (4) areas where methods are common to forensic analysis and research in contraceptive use and monitoring. Information and progress made in these areas coincide with the research interests of forensic biology and cross-talk between these disciplines may benefit both.     

肝素对TNBS诱导的大鼠结肠炎的治疗作用及其机制

背景:活动期炎症性肠病(IBD)往往伴有高凝状态,高凝状态与炎症之间的关系尚未完全明确。目的:探讨三硝基苯磺酸(TNBS)诱导的大鼠结肠炎模型中凝血异常与炎症的关系以及肝素的治疗作用及其机制。方法:40只Sprague-Dawley大鼠分为正常对照组、结肠炎组、肝素治疗组和肝素+鱼精蛋白组。后三组以TNBS诱导结肠炎模型,行疾病活动指数(DAI)、大体和组织病理学评分,检测血清肿瘤坏死因子(TNF)-α水平、凝血酶原时间(PT)、激活部分促凝血酶原激酶时间(APTT)、抗凝血酶(AT)活性和血小板计数。结果:与正常对照组相比,结肠炎组DAI、大体评分和组织病理学评分以及血清TNF-α水平显著增高(P0.05),PT、APTT、AT活性均显著降低(P0.05),血小板计数显著增高(P0.05)。经肝素治疗后,上述指标均显著改善(P0.05)。肝素+鱼精蛋白组各指标与结肠炎组无明显差异。结论:TNBS诱导的结肠炎模型中存在凝血异常,肝素可有效控制炎症激活的凝血异常,其抗炎机制可能与抗凝活性相关。鱼精蛋白可中和肝素的抗凝活性同时终止其抗炎作用。     

Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines

Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A(3) (CMA(3)). Increases in DFI (15%), DFI% (4.5-fold), and CMA(3) (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA(3) provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.     

鱼精蛋白对牛主动脉平滑肌细胞增殖的影响

应用细胞培养技术进行新生牛主动脉平滑肌细胞传代培养，取第４～６代平滑肌细胞进行实验，采用细胞计数法观察不同浓度的鱼精蛋白对牛主动脉平滑肌细胞增殖的影响。结果显示：不同浓度鱼精蛋白均能刺激牛主动脉平滑肌细胞增殖，鱼精蛋白的浓度与平滑肌细胞增殖量呈正相关。上述结果提示：鱼精蛋白通过促进动脉平滑肌细胞增殖，参与动脉粥样硬化的发生和发展     

TGF-βⅡR单链抗体/轻链恒定区/鱼精蛋白截短体融合蛋白携带miR-29b抗肝纤维化的研究

目的 观察TGF-βⅡR单链抗体/轻链恒定区/鱼精蛋白截短体融合蛋白(简称新型载体)携带miR-29b对体外培养肝星状细胞(HSC)的转染效率和治疗效果,为今后肝纤维化基因治疗提供新载体.方法 脂质体、新型载体和慢病毒携带miR-29b治疗体外培养的HSC,荧光显微镜和流式细胞术观察转染效率,实时荧光定量RT-PCR和Western Blot技术分析collagen α(1) (Ⅰ) mRNA和蛋白表达水平.结果 与对照组比较,慢病毒组的转染效率最高,为70％;其次为新型载体组,为58％;最后为脂质体组,为29％.各载体组collagen α1(Ⅰ) mRNA表现出不同的下降,与对照组比较,慢病毒组下降70％(t=6.316,P＜0.01),新型载体组为50％(t=4.082,P＜0.01),脂质体组为38％(t =3.014,P＜0.05).各载体组collagen α1 (Ⅰ)蛋白也表现出不同的下降,慢病毒组下降为59％(t=4.209,P＜0.01);新型载体组为41％(t =4.033,P＜0.01;);脂质体组为27％(f=2.842,P＜0.05).结论 笔者构建的新型载体具有较高的转染效率,并且携带miR-29b具有较好的抗肝纤维化效果.     

酸性环境对先天性心脏病患儿体外循环术后鱼精蛋白中和作用的影响

目的探讨先天心性脏病术后患儿不同酸碱度对鱼精蛋白中和肝素的影响。方法回顾2011年3月至2011年4月间对108例先天性心脏病患儿行体外循环下行心脏直视手术的病例资料,分析其临床特点及不同血浆酸碱度时鱼精蛋白用量对术后引流的影响。结果在酸性环境下,鱼精蛋白中和肝素剂量及术后引流量[（136．8±22．8）m1]高于对照组[（112．6±22．7）m1],差异有统计学意义（P〈0．01）。在非酸性环境下,鱼精蛋白和肝素比平均值为1．23：1;在pH〈7．30或剩余碱（BE）〈-6时,鱼精蛋白的用量显著增加,鱼精蛋白和肝素比平均值为1．86：1。结论不同的酸碱度对鱼精蛋白中和肝素有影响,酸性环境下鱼精蛋白的用量增加,术后引流量也会增多。在体外循环结束时,测得pH〈7．30或BE〈-6时,建议纠正酸碱平衡后按剂量使用加鱼精蛋白中和肝素。     

颈动脉内膜切除术中应用鱼精蛋白中和肝素的临床观察

目的 评价颈动脉内膜切除术中应用鱼精蛋白中和肝素对术中止血时间、术后引流量及相关并发症的影响.方法 收集2012年1至12月于北京协和医院行颈动脉内膜切除术治疗的125例次颈动脉狭窄患者的病例资料,进行回顾性队列研究.根据术中是否使用鱼精蛋白,将患者分为鱼精蛋白组和非鱼精蛋白组.分析和比较两组的病例资料、术中止血时间、术后伤口引流量及相关并发症.结果 125例次颈动脉内膜切除术中鱼精蛋白组共75例次,非鱼精蛋白组共50例次.鱼精蛋白组术后第1天伤口引流量少于非鱼精蛋白组,分别为(14±13)ml和(19±12)ml,P=0.038;鱼精蛋白组术后总引流量平均值小于非鱼精蛋白组,分别为(20±17)ml和(26±15)ml,P=0.035;鱼精蛋白组术中止血时间较非鱼精蛋白组缩短,分别为(30±11)min和(36±11) min,P=0.030.125例次手术无一例因手术死亡,其中1例(0.8％)发生切口血肿,急诊行血肿清除后恢复良好,1例(0.8％)患者发生术后脑卒中加重,经内科保守治疗后好转.结论 颈动脉内膜切除术中选择性使用鱼精蛋白,对缩短术中止血时间、减少术后伤口引流量有积极作用,且不增加缺血性脑卒中的风险.     

升主动脉注射鱼精蛋白对体外循环后婴儿细胞因子与补体的影响

目的 通过升主动脉及上腔静脉注射鱼精蛋白,观察和评价不同注射途径对体外循环后婴儿血浆IL-6、TNF-α、G3a、G5a浓度的影响.方法 年龄≤12个月行体外循环心内直视手术的患儿60例,数字随机分为实验组(经升主动脉注射鱼精蛋白组,n=30)和对照组(经上腔静脉注射鱼精蛋白组,n=30).于注射鱼精蛋白前(T1)、注射鱼精蛋白后1 h(T7)取右心房肝素抗凝血1 ml,用ELISA法测定血浆IL-6、TNF-α、G3a、C5a浓度.分别于注射鱼精蛋白前、注射鱼精蛋白后1 min、2min、3 min、5 min、10min测左侧桡动脉动脉收缩压.结果 注射鱼精蛋白前两组患儿血浆IL-6、TNF-α、C3a、C5a浓度无统计学差异(P＞0.05);对照组患儿在注射鱼精蛋白后1 h血浆IL-6浓度(25.4±10.7)ng/L、TNF-α浓度(26.1±0.2)ng/L、C3a浓度(23.0±3.5)μg/L、G5a浓度(0.9±0.1)μg/L,均较注射鱼精蛋白前显著增多(P＜0.01);实验组患儿在注射鱼精蛋白后1h血浆TNF-α浓度(25.2±0.4)ng/L、C3a浓度(19.6±3.5)μg/L、C5a浓度(0.8±0.1)μg/L均较注射鱼精蛋白前无显著性差异(P＞0.05);IL-6浓度均较注射鱼精蛋白前有统计学差异(19.2±10.2)ng/L(P＜0.05);对照组患儿在注射鱼精蛋白后1 h血浆IL-6、TNF-α、C3a、C5a浓度较实验组显著增高(P＜0.01).注射鱼精蛋白前两组患儿左侧桡动脉收缩压分别为(90.9±4.9)mmHg、(89.8±6.0)mmHg无统计学差异(P＞0.05);注射鱼精蛋白后1 min、2 min、3 min两组患儿左侧桡动脉收缩压分别为(86.3±4.7)mm-Hg、(85.1±5.4)mmHg、(79.2±4.4)mmHg、(80.1±5.1)mmHg、(82.5±4.5)mmHg、(84.1±4.5)mmHg,较注射鱼精蛋白前均下降(P＜0.01);两组患儿同时点左侧桡动脉收缩压相互比较无统计学差异(P＞0.05).结论 升主动脉途径注射鱼精蛋白可显著减少体外循环后婴儿血浆IL-6、TNF-α、G3a、G5a浓度.

Abstract：

Objective To study the effects of protamine on complement activation and cytokine generation in infants with cardiopulmonary bypass. Methods Sixty infants under 1 year of age underwent cardiopulmonary bypass (CPB) from heart surgery were recruited in this study, and randomly grouped into 2 groups. Experimental group had 30 patients, who received protamine injection via ascending aorta. The control group also had 30 patients, but they received protamine injection via superior vena cava. Blood samples were collected from right atrium at the time prior to protamine injection (T1) and 1 hour after the injection (T2). IL-6, TNF-α, C3a and Csa plasma levels were measured by ELISA. Left radial artery systolic arterial pressures (SAPs) were measured at the time prior to protamine injection (Time 1), 1 min (Time 2), 2 min (Time 3), 3 min (Time 4), 5 min (Time 5), and 10min (Time 6) after the injection. Results At Time 1, IL-6, TNF-α, C3a and C5a plasma levels were not significantly different between the control group and the experimental group (P＞0. 05). In the patients of control group, IL-6 (25. 4 ± 10. 7 ng/L), TNF-α (22. 1 3. 5μg/l), C3a (23. 0 ± 3. 5 μg/L), and C5a (0. 9 ± 0. 1 μg/L) at Time 7 were significantly increased compared with these at Time 1 (P＜0. 01). In the patients of experimental group, levels of TNF-α (25. 2 ± 0. 4 ng/L), C3a (19. 6 ± 3. 5μg/L) and Csa (0. 8 ± 0. 1 μg/L) at Time 7 were not significantly different from those at Time 1 (P＞0. 05), but IL-6 (19. 2± 10. 2 ng/L) at Time 7 was significantly increased than that at Time 1 (P＜0. 05). Before injection, no difference of left radial artery SAPs was found between experimental group and control group (P＜0. 05). After injection, the left radial artery SAPs of the patients of the 2 groups were decreased compared with the SAPs before injection (P＜0. 05), but no difference between experimental and control group was found (P＞0. 05). Conclusions Protamine injection via ascending aorta decreased IL-6, TNF-α, C3a and C5a plasma levels in the infants with cardiopulmonary bypass.