成骨细胞中Runx2对机械离心力刺激的响应

目的研究机械离心力刺激对MC3T3-E1细胞中Runx2表达的影响,探讨成骨细胞将力学刺激转化为生化响应过程中骨形态发生蛋白（BMP）信号转导通路的作用。方法将MC3T3-E1细胞分成A、B、C、D组,分别使用含10%胎牛血清、10%胎牛血清、100 ng·mL-1 Noggin和100 ng·mL-1 Noggin的DEME培养基预处理24 h,对B、D组加载271×g离心力5 min,A、C组不进行离心加力,其余条件相同。30 min后4组分别同时收获细胞,提取总RNA,并逆转录为cDNA,通过实时荧光定量PCR检测Runx2基因表达情况。结果B组Runx2 mRNA表达较A组明显增高（P<0.05）,D组Runx2 mRNA表达比B组表达明显降低（P<0.05）,A、C、D组间表达水平比较差异无统计学意义（P=0.692）。结论BMP信号转导通路参与成骨细胞对机械离心力刺激的响应过程,并可能在成骨细胞对力学信号引起的信息传递级联反应中扮演重要角色。     

HER2高表达的人乳腺癌原代细胞模型的建立

目的:探讨人乳腺癌恶性胸水肿瘤细胞的分离、纯化、培养与肿瘤细胞的鉴定,为HER2阳性的人乳腺癌的研究提供肿瘤原代细胞模型.方法:利用淋巴细胞分离液及密度梯度离心法分离、纯化恶性胸水肿瘤细胞,分离得到的细胞培养并传至第5代,通过流式细胞仪分选肿瘤细胞、Q-PCR技术、Western blot技术验证细胞HER2分子的表达水平、通过裸鼠荷瘤实验检测细胞的成瘤能力.结果:淋巴细胞分离液及密度梯度离心法分离得到乳腺癌原代细胞;流式细胞分选结果显示未分选出HER2高表达的肿瘤细胞;Q-PCR及Western blot检测发现分离得到的细胞在mRNA及蛋白水平HER2的表达水平高于乳腺癌MCF-7细胞;裸鼠荷瘤实验结果显示分离得到的细胞可以成功荷瘤.结论:胸水中分离得到的细胞为HER2阳性的乳腺癌原代细胞;对于原代细胞的鉴定需要采用不同的方法加以论证.     

Dissociation and re-association studies on the interaction domains of mannan-binding lectin (MBL)-associated serine proteases,MASP-1 and MASP-2, provide evidence for heterodimer formation

Activation of the lectin pathway of complement begins with the activation of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, which are bound to the recognition molecules, MBL and ficolins. MASPs are Ca²⁺-dependent dimers. Dimerization and Ca²⁺-dependent association with the recognition molecules occurs via the first 3 domains, the CUB1-EGF-CUB2 region. The CUB1-EGF-CUB2 (D1-3) regions of MASP-1 and MASP-2, and also their tagged versions, were expressed in E. coli, refolded and purified. The first three domains of MASP-1 are identical with the respective regions of MASP-3 and MAp44, which are also associated with MBL and ficolins. The functionality of the fragments was checked by inhibition of C3 deposition from human serum. Time-course of the dissociation and re-association was examined by size exclusion chromatography. Both refolded proteins are tight Ca²⁺-dependent dimers, as expected. In buffer containing EDTA MASP-1_D1-3 dissociated to monomers, however it took about 1 h to reach an equilibrium. Upon re-calcification dimers were re-formed, but this process was even slower; only after overnight incubation was the dimerization completed. MASP-2_D1-3 showed a somewhat different behavior: dissociation by EDTA was even slower, less complete, and higher MW aggregates also appeared. Heterodimer formation was detected by native PAGE. As modeled by the D1-3 fragments, MASP-1 and MASP-2 can readily form heterodimers after dissociation and re-association, however, in the presence of Ca²⁺ exchange of subunits is slow between the homodimers. MASP-1:MASP-3 heterodimer formation was modeled by the tagged and untagged D1-3 fragments, and data indicate that subunits of these proteins are readily exchanged even in the presence of Ca²⁺. The existence of heterodimers influences the current view on the composition of lectin pathway complexes and their activation.     

雅宁片联合密度梯度离心制备法治疗少、弱精子症

目的探索男性不育患者中少、弱精子症的联合治疗方法。方法本中心2005年4月~2006年4月共210例少、弱精子症患者,分为3组。即经密度梯度离心制备后对其配偶行宫腔内人工授精(IUI)组70例(对照组1),雅宁片治疗组68例(对照组2),雅宁片治疗后联合密度梯度离心制备法对其配偶行IUI治疗组72例(实验组)。检测治疗前、后精液常规,并随访3组患者治疗期间配偶妊娠结局。结果雅宁片治疗前、后2组精子密度,精子活动率比较,有显著性差异(P<0.05)。实验组雅宁片治疗后联合密度梯度离心制备法对其配偶行IUI后,配偶妊娠结果与对照组比较,有显著性差异(P<0.05)。结论雅宁片联合密度梯度离心制备法是治疗男性不育患者中少、弱精子症有效的联合治疗方法。     

Assessment of density gradient centrifugation (DGC) and sperm chromatin dispersion (SCD) measurements in couples with male factor infertility undergoing ICSI

Purpose

To investigate how effectively density gradient centrifugation (DGC) improves sperm nuclear integrity and to determine whether the sperm chromatin dispersion (SCD) test of sperm nuclear integrity in native or DGC-treated semen can predict the outcome of assisted reproductive technology (ART) in couples undergoing intracytoplasmic sperm injection (ICSI).

Methods

The DNA integrity of spermatozoa from 63 male factor infertility patients undergoing ICSI was analyzed by the SCD test before and after DGC. The predictive value of the sperm DNA fragmentation index (DFI) for ART outcomes was assessed in a cohort of 45 patients who were undergoing fresh embryo transfer. For the analysis, they were divided into pregnant and non-pregnant groups and, independently, into high sperm DFI (DFI > 30 %) and low sperm DFI (DFI ≤ 30 %) groups. Both raw and DGC semen parameters were examined.

Results

In the asthenospermia and oligozoospermia groups, DGC decreased the sperm DFI from 31.5 ± 19.7 and 28.5 ± 10.3 to 19.2 ± 18.3 and 16.0 ± 12.8, respectively (P < 0.01). DGC decreased the sperm DFI in the severe oligozoospermia group from 41.4 ± 19.0 to 36.3 ± 20.6 (P > 0.01). The pregnant and non-pregnant groups did not differ in their fertilization rate and sperm DFI in native or DGC semen (P > 0.05). There was also no significant difference between the high sperm DFI (DFI > 30 %) and low sperm DFI (DFI ≤ 30 %) groups with regard to fertilization rate, implantation rate, and clinical pregnancy rate for both native and DGC semen (P > 0.05). The patients undergoing ICSI with a high sperm DFI had a higher pregnancy loss rate (defined as spontaneous miscarriage or biochemical pregnancy) compared with patients with a low sperm DFI in both the native and DGC semen groups.

Conclusions

DGC highly significantly reduces sperm DNA fragmentation in the semen of ICSI patients, with the exception of those with severe oligozoospermia. The results of the SCD test of sperm DNA fragmentation in native or DGC semen do not correlate with the fertilization rate, implantation rate, or clinical pregnancy rate in patients undergoing ICSI.     

大鼠MSCs的体外分离、培养及鉴定的实验研究

[目的]体外分离、培养及鉴定大鼠骨髓间充质干细胞（MSCs）。[方法]密度梯度离心法体外分离、培养7周龄sD大鼠MSCs,利用差速贴壁原理纯化MSCs,并通过形态学观察、细胞表面抗原标志物、多细胞系诱导分化对其进行鉴定。[结果]第3代（P3代）不表达抗原CD34,表达抗原CD44,符合MSCs表面标志物特征。[结论]密度梯度离心法能够建立稳定的MSCs体外分离培养体系。     

中成药生产中离心薄膜浓缩技术的初步研究

在中成药生产中，采用离心薄膜蒸发器浓缩药液，通过3只产品正交试验，以蒸发量，浓缩比和产品的主要成分含量为考核指标，求得最佳工艺，从而摸索其普遍规律。     

兔骨髓间充质干细胞的分离、培养与鉴定

目的 优化获取兔骨髓间充质干细胞并对其进行纯化、鉴定,同时对其生物学性状进行研究。方法 取新生新西兰大耳白兔骨髓5．0mL,采用密度梯度离心法、贴壁筛选法并结合传代对其进行纯化,观察细胞形态、超微结构和表面标志物的表达,从而对其进行鉴定。观察第1、3、5、8、10代细胞的增殖情况,并绘制出生长曲线。结果 经密度梯度离心、贴壁筛选并结合传代所得到的间充质干细胞很纯,第3代和第5代细胞形态单一,细胞排列具有典型的漩涡状结构。第1、3、5代细胞增殖能力强,生长旺盛;而第8、10代细胞增殖明显减弱。所分离培养的细胞表达CD44、CD90,不表达CD34,电镜观察为低分化细胞。结论 分离培养的细胞为间充质干细胞且成分单一,具有干细胞特性．第3、5代细胞很纯且其增殖能力强,适用于做以后的研究。     

提高中药口服液澄明度方法的研究进展

中药口服液属于中成药液体制剂,澄明度好的口服液一般内在质量也比较优良。为了更好地控制中药口服液的质量,需要在制剂工艺中去除杂质以提高澄明度。目前提高中药口服液澄明度的方法主要有醇沉法、高速离心法、膜分离法、大孔吸附树脂法、澄清剂吸附法、明胶法、调节pH值法等。综述了近年来常用的改善中药口服液澄明度的新技术、新工艺及其在生产中的应用情况。     

高速离心法和稀释法处理乳糜血对光学法凝血指标检测结果的影响

目的 探讨高速离心法和稀释法处理乳糜血对光学法凝血指标检测结果的影响.方法 以我院检验科2013年4月至2014年4月接收60例正常凝血标本与60例乳糜血凝血标本为对象.正常凝血标本组采用稀释法对PT、APTT和FIB进行检测并对回归方程加以建立.乳糜血凝血标本组根据三酞甘油浓度不同,分为轻度、中度、重度三组;在PT、APTT和FIB的检测上,对稀释法和高速离心法加以采用;同时在回归方程中对稀释后的检测结果加以代人,比较代入后所得结果与高速离心法结果.结果 回归分析正常凝血标本的原血浆与稀释血浆PT、APTT和FIB的检测结果,分别得出PT、APTT和FIB的回归方程(稀释前与稀释后结果分别用Y与X表示).PT:Y=0.305X+4.29,APTT:Y=0.155X+22.607,FIB:Y=2.883X+0.218.PT、APTT和FIB回归方程中r分别为0.860、0.155、0.821,P均为0.000.稀释法与高速离心法对PT、APTT和FIB的检测中,其结果在轻度、中度乳糜血组差异无统计学意义(P＞0.05);在重度乳糜血组检测结果中,稀释法中PT的检测结果及FIB的含量明显比高速离心法高(P＜0.05).结论 当在乳糜血标本的凝血项目检测中应用光学法仪器时,高速离心法比稀释法具有更强的抗干扰能力,适用范围更广,结果也更加准确,更有利于出血性疾病诊断.