细胞图案化研究进展

细胞图案化是一种研究和控制细胞行为的有效实验方法, 广泛用于细胞生物学、组织工程学、药物筛选和创伤治疗等各个研究领域. 介绍了各种细胞图案化的制备方法, 并对细胞图案化中涉及的特征物质以及有关动态基底作了简要评述.     

Isolation and Purification of Polysaccharides from Cordyceps minlitaris and Its Inhibition on the Proliferation of Rat Glomerular Mesangial Cells

The crude polysaccharide was obtained by means of the decolorization of porphyrized Cordyceps minlitaris stroma with organic solvent, extraction with hot water, precipitation in 80% ethanol, and protein removal with the Sevag method. After purification with Sephadex G-75, two of its components, CMP-1 and CMP-2, were obtained. Through the assay of gel chromatography and polarimetry, CMP-1 was identified as pure polysaccharide. The results demonstrated that CMP-1 had favorable oxidation resistance activity, which could scavenge not only oxygen-free radicals in the self-oxidation system of pyrogallic acid, but also the hydroxide-free radicals in the Fenton system. The study focused on the effects of low, medium, and high dosages of CMP-1 in rat blood serum on the proliferation of glomerular mesangial cells in vitro. Through MTT Colorimetric analysis, the activities were compared among the blank control group and the Niaoduqing positive control group CMP-1 and CMP-2. The results shows that CMP-1 was able to inhibit the proliferation of rat glomerular mesangial cells effectively. Therefore, CMP-1, one component of polysaccharides of Cordyceps minlitaris, was certainly a potential remedy for hyperplastic glomerular nephritis, whose antioxidant activity could slow down the process of chronic renal failure（CRF） to some extent.     

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.     

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells ls174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells Is174t and the growth of colon cancer tissue of tumor-bearing mice,MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro.Transmission electron microscope was used to observe the cell apoptosis.FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle,RT-PCR method was used to detect the expressions of bc1-2 and bax mRNA.Western blot method was used to detect the expressions of bc1-2 and bax protein.Cells isi74t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor.RT-PCR method was used to detect the expressions of bc1-2 and bax mRNA in the tumor tissue.Western blot method was used to detect the expressions of bc1-2 and bax protein in the tumor tissue.Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro(P<0.01).It is able to induce the apoptosis of cells Is174t,causing the decrease in the expression of bc1-2 and the increase in the expression of bax.Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice(P<0.05),causing the decrease in the expression of bc1-2 and the increase in the expression of bax.Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor.The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bc1-2/bax expression.     

胞外囊泡异质性无标记表征技术研究进展

胞外囊泡(extracellular vesicles, EVs)是细胞在信息传递和环境调控时产生的磷脂双分子层膜包裹的囊泡(尺寸为30～1000 nm).近十五年,其生物及临床相关研究发展迅猛,已被视为是细胞间信息传递、癌宿主调节等动态过程的关键因素之一.尽管胞外囊泡具有重要的生物学功能,但其尺寸小、结构脆弱且易聚集,传统生化分析和检测技术难以对其实现精准表征.目前, EVs相关的大多数表征技术采用“批量分析”形式,仅提供大量外泌体的平均结果.为了加深对胞外囊泡异质性的认识,以单个EVs为目标的表征技术将成为新的趋势.本文概述了单颗粒水平胞外囊泡表征的光学和非光学的无标记表征方法,包括拉曼显微、等离激元技术、原子力显微和电子显微技术等.相比于传统整体分析方法,单颗粒水平胞外囊泡表征能够提供更加丰富的信息,在生物医药基础研究领域有着广阔前景.     

应用不同光谱技术,动物RBC,Hb及人皮肤表面血流氧化-还原状态检测

目的:检测与分析实验动物血液RBC(RBC·O2,RBC·CO2),Hb(HbO2,HbCO2)和人体皮肤表面流动血液氧化·还原状态的成像与未成像可见光谱领域OD值特征,并为该技术对白癜风病表皮黑色素颗粒检测中的应用尊定基础。方法:利用不同光谱技术和invitro和invivo检测手段,统计分析血液不同状态下波长与位置的OD值信息。结果:invitro检测:动物血液Hb·O2和RBC·O2两者在可见领域均有367,414(Soret带)nm与541,576(Q带)nm的吸收峰位;血液Hb·CO2和RBC·CO2均有432(Soret带)与和553(Q带)nm的波长吸收峰位;血液RBC状态和Hb溶血状态波长吸收峰位无改变,只是在氧化与还原状态下有完全独立的吸收峰位,血液RBC状态和Hb溶血状态波长吸光度OD值之间,有显著性差异(p<0·01)。浓度为1·5×107cell·mL-1的RBC·O2和Hb·O2在576nm的吸收峰位吸光度(y)与红细胞浓度(x)做成两条回归曲线:既,Hb·O2(b1)^y=0·05 0·983x;RBC·O2(b2)y^=0·127 1·934x,两者之间差异有显著性(p<0·01)。invivo检测:在人手背皮肤表面ImSpector图像中RBC·O2状态在540,576nm,RBC·CO2状态在555和755nm处有吸收峰。选择(a:指甲,b:指,c:手背)三个点位分别进行波长检测,每点(n=10)545nm吸收峰的平均OD值,依次为0·83±0·001,0·73±0·001和0·62±0·001,其三处测定点的OD值之间有显著性差异(p<0·01)。结论:invitro检测的RBC与Hb两者波长吸收峰位不变,但吸光度OD值不同,认为RBC状态测定结果更接近于活体组织血管内原始状态。invivo检测对人体无任何侵袭与损伤,灵敏度高,测试时间短,并且同时获得被测样品的波长与位置信息画面等优势,有望表皮中黑素等有色颗粒的直接检测。     

新型生物黏附性材料巯基壳聚糖的合成与表征

巯基聚合物利用二硫键的形成, 以共价键黏附于黏膜表面, 可以延长药物在黏膜上的滞留时间, 有利于药物分子的吸收. 本文合成了一种新型巯基聚合物——N-乙酰基-L-半胱氨酸-g-壳聚糖(CS-NAC), 并进行了表征, 同时对材料的溶胀度、黏附性和细胞毒性进行了测试. 结果表明, 这种巯基聚合物具有较高的巯基含量, 最高可达到589.3 μmol/g; 具有快速的溶胀性能; 黏附性显著增强, 黏附时间和黏附力分别是壳聚糖的30和3倍; 无明显的细胞毒性. 因此, CS-NAC是一种很有应用前景的生物黏附性材料.     

乙二醛诱导细胞凋亡的近红外表面增强拉曼光谱

将60 nm金纳米粒子导入到活的人骨肉瘤细胞中, 用近红外表面增强拉曼散射(SERS)技术获取细胞内化学成分的高分辨SERS信息. 对正常活性细胞和乙二醛诱导的凋亡细胞的比较研究表明, 对于正常活性的细胞, 金纳米探针主要分布在细胞质内(围绕细胞核), 而凋亡细胞内的金纳米探针的分布较为均匀, 在遍布凋亡细胞内的各个位置包括细胞表面均容易找到DNA片段的信息.     

大肠杆菌HB101感受态细胞的显微观察与分析

对数生长前期的大肠杆菌在生理盐水中用CaCl2可以直接诱导建立感受态并完成转化过程。利用H－8100透射电子显微镜观察用CaCl2直接在生理盐水中诱导建立感受态的大肠杆菌细胞，发现感受态细胞整体电子密度趋于均匀，但存在有电子密度较高颗粒，表明大肠杆菌感受态建立过程中，细胞整体对电子通透性增强，可能存在涉及细胞膜通透性的复合物重新形成相关的胞内物质代谢及其在细胞内定位的受控改变，暗示大肠杆菌可能具有建立自然感受态的能力。     

一株木麻黄弗兰克氏菌的细胞化学组分

采用薄层层析和氨基酸分析仪检测了细枝木麻黄弗兰克氏菌菌株Ｃｃ０１的细胞化学组分．表明它的全细胞特征性糖为木糖和半乳糖；细胞壁氨基酸组分在薄层上只显示有赖氨酸．而无ｍｅｓｏ－ＤＡＰ和甘氨酸；但在氨基酸分析仪上除含有谷氨酸（ＧＬＵ）、甘氨酸（ＧＬＹ）、丙氨酸（ＡＬＡ）和二氨基庚二酸（ＤＡＰ）以外，尚有较高含量的天冬氨酸（ＡＳＰ）和赖氨酸（ＬＹＳ）．由这些氨基酸的分子比得出，菌株Ｃｃ０１与胞壁Ⅱ型和Ⅲ型的参考菌株均明显不同，其特征性氨基酸分子比为Ｇｌｕ∶Ｇｌｙ∶Ａｌａ∶Ｄａｐ∶Ａｓｐ∶Ｌｙｓ为１．００∶０．１３∶３．５０∶０．２１∶０．５３∶０．７８，其ＤＡＰ含量仅为Ⅲ型菌株的１／６．表明Ｃｃ０１的胞壁类型和其它弗兰克氏菌不一样，是一种特殊类型．