两种体外细胞毒性检测方法的比较研究

目的比较两种常用的细胞毒性检测方法在医疗器械生物学评价中的相关性。方法分别采用MTT比色法和细胞增殖度法,在37℃条件下,将五种医疗器械／生物材料的浸提液分别与小鼠成纤维细胞（L-929）接触2天和2,4,7天,比较材料对细胞的毒性影响。结果5种不同的材料浸提液分别表现出不同程度的细胞毒性反应（0～2级）。将MTT比色法与细胞增殖度法（2天）的实验数据进行相关性分析,显示两者之间具有良好的相关性（R=0．977）。结论MTT比色法由于其检测所需的细胞量相对较少,试验步骤相对简便、检测周期短,因此具有一定的优越性,是个值得推荐的细胞毒性检测方法。     

siRNA抑制S100A4 表达对卵巢癌细胞CP70生物学特性的影响

摘要 目的：探讨人卵巢癌顺铂耐药细胞株CP70沉默S100A4 基因后,CP70细胞对顺铂敏感性、凋亡及细胞迁移的影响。方法：设计并合成S100A4基因特异性的siRNA并转染入卵巢癌细胞CP70,48 h后应用RT-PCR和Western Blot检测在mRNA和蛋白水平siRNA对S100A4的影响,MTT法检测转染 siRNA后卵巢癌细胞CP70对顺铂敏感性的变化。;用流式细胞术检测顺铂（40 μM） 对转染S100A4 siRNA后对卵巢癌细胞CP70凋亡的影响,Transwell法观察siRNA抑制S100A4后对卵巢癌CP70迁移能力的影响。 结果：与空白对照组、阴性对照组相比,S100A4 siRNA转染组CP70细胞的S100A4基因和蛋白表达降低(P<0.01)。MTT法检测顺铂敏感性发现S100A4 siRNA转染组CP70细胞顺铂敏感性增强。在顺铂刺激下,siRNA转染组细胞凋亡率高于其他各组,差异具有统计学意义（P<0.05）。Transwell发现CP70细胞迁移能力明显下降（P<0.05）。结论：S100A4 siRNA能够明显抑制CP70细胞S100A4的表达,从而增强细胞对顺铂的敏感性,促进细胞凋亡,减弱细胞的迁移能力。S100A4有望成为逆转卵巢癌铂类耐药的治疗靶点。     

siRNA抑制S100A4表达对卵巢癌细胞CP70生物学特性的影响

目的：探讨人卵巢癌顺铂耐药细胞株CP70沉默S100A4基因后,CP70细胞对顺铂敏感性、凋亡及细胞迁移的影响。方法：设计并合成S100A4基因特异性的siRNA并转染入卵巢癌细胞CP70,48 h后应用RT-PCR和Western Blot检测在mRNA和蛋白水平siRNA对S100A4的影响,MTT法检测转染siRNA后卵巢癌细胞CP70对顺铂敏感性的变化。用流式细胞术检测顺铂（40μM）对转染S100A4 siRNA后对卵巢癌细胞CP70凋亡的影响,Transwell法观察siRNA抑制S100A4后对卵巢癌CP70迁移能力的影响。结果：与空白对照组、阴性对照组相比,S100A4siRNA转染组CP70细胞的S100A4基因和蛋白表达降低（P〈0.01）。MTT法检测顺铂敏感性发现S100A4 siRNA转染组CP70细胞顺铂敏感性增强。在顺铂刺激下,siRNA转染组细胞凋亡率高于其他各组,差异具有统计学意义（P〈0.05）。Transwell发现CP70细胞迁移能力明显下降（P〈0.05）。结论：S100A4 siRNA能够明显抑制CP70细胞S100A4的表达,从而增强细胞对顺铂的敏感性,促进细胞凋亡,减弱细胞的迁移能力。S100A4有望成为逆转卵巢癌铂类耐药的治疗靶点。     

冻融联合灌注法优化大鼠肾脏脱细胞支架的制备

本研究旨在探索优化肾脏脱细胞支架的制备方法,为肾脏组织工程及肾脏体外病理、毒理研究提供实验基础。取大鼠肾脏灌注PBS作为对照组 (Control组),在不同流速下分别以十二烷基磺酸钠 (Sodium dodecyl sulfate,SDS) 灌注 (S组),Triton X-100联合SDS灌注 (TS组),反复冻融后Triton X-100联合SDS灌注(FTS组),制备肾脏脱细胞支架,并测定其流体分布及脉管阻力。HE染色、DAPI染色、DNA定量检测脱细胞支架脱细胞程度,Masson染色、PAS染色、免疫组织化学染色检测脱细胞支架主要成分的保留和结构的完整,扫描电镜检测支架的超微结构,MTT法检测支架的细胞毒性,ELISA检测支架中生长因子的含量。结果显示,FTS组脱细胞用时较S组、TS组少,10 mL/min组支架脉管阻力较低,S组、TS组、FTS组流体分布与Control组存在差异。HE染色和DAPI染色显示各组支架未见细胞成分残留,DNA含量＜50 ng/mg。Masson染色和PAS染色可见细胞外网状胶原及多糖,免疫组织化学染色见Ⅰ型胶原 (CollagenⅠ)、Ⅳ型胶原蛋白 (Collagen Ⅳ)、纤维连接蛋白 (Fibronectin)、层粘连蛋白 (Laminin) 表达。扫描电镜见支架呈蜂窝状结构。MTT法检测支架细胞毒性分级在0–1级之间。ELISA检测提示FTS组VEGF、EGF、IGF-1、PDGF含量明显高于S组和TS组。综上,联合冻融和灌注法能够制备更为理想且有效的大鼠肾脏整器官脱细胞支架,为肾脏组织工程及肾脏体外病理、毒理学研究奠定基础。     

紫球藻胞外多糖抗柯萨奇B3病毒活性研究Ⅰ

采用MTT法研究了紫球藻胞外磺酸化多糖(ESPS)的细胞毒性,在高浓度(250μg/mL)下尚未发现对HEp-2细胞有明显毒性.CPE抑制法发现ESPS能明显抑制细胞的CPE,在综合、预防、治疗和直接作用四种给药方式下都具有抑制CVB3的作用.直接作用法的活性最高,TI值高达1920.6,是阳性对照药病毒唑的31倍.ESPS组分中分子量越大,抗病毒活性越高,其中分子量大于300 k的组分,IC50可低至0.754μg/mL,TI值高达331.6,是病毒唑的5倍多.结果表明ESPS有强烈的抗CVB3病毒活性,作用机制可能是干扰病毒在细胞表面的吸附及侵染过程.     

紫球藻胞外多糖抗柯萨奇B3病毒活性研究I

采用MTT法研究了紫球藻胞外磺酸化多糖(ESPS)的细胞毒性,在高浓度(250μg/ml)下尚未发现对HEp-2细胞有明显毒性。CPE抑制法发现ESPS能明显抑制细胞的CPE,在综合、预防、治疗和直接作用四种给药方式下都具有抑制CVB3的作用。直接作用法的活性最高,TI值高达1920．6,是阳性对照药病毒唑的31倍。ES-PS组分中分子量越大,抗病毒活性越高,其中分子量大于300k的组分,IC50可低至0．754μg／ml,TI值高达331．6,是病毒唑的5倍多。结果表明ESPS有强烈的抗CVEb病毒活性,作用机制可能是干扰病毒在细胞表面的吸附及侵染过程。     

CPE对体外培养前列腺癌细胞株的抑制作用

目的:探讨产气荚膜梭菌肠毒素(CPE)对前列腺癌细胞株在体外培养过程中的抑制作用。方法:以细胞免疫荧光染色观察人前列腺癌PC-3细胞株和22RV1细胞株表达Claudin-3情况;并分别用生长曲线测定法及MTT法等测定CPE对PC-3和22RV1细胞活性的影响;分析CPE对肿瘤的生长抑制作用。结果:应用Claudin-3单抗进行免疫荧光染色,证实体外培养的人前列腺癌细胞株22RV1可以特异性高表达Claudin-3,而在PC-3上有弱表达;用相关方法测定经过不同浓度的CPE处理后的前列腺癌细胞株22RV1细胞增殖明显降低,有明显抑制作用;而对PC-3则未产生明显细胞毒性作用。结论:CPE在体外可通过特异性作用于Claudin-3高表达的前列腺癌细胞株对前列腺癌细胞产生抑制作用。     

S100A9促进肝癌细胞HepG2的存活与侵袭依赖于RAGE

目的:探讨S100A9对人肝癌细胞系HepG2生物学行为的影响及可能机制。方法:采用免疫组织化学法与Western blot方法检测人肝癌组织与癌旁组织中S100A9蛋白表达水平;原核表达重组蛋白的方法构建重组蛋白GST-S100A9,用GST-S100A9处理肝癌细胞HepG2和肝正常细胞L02,然后用MTT法检测细胞存活能力,Transwell侵袭实验检测细胞侵袭力;Western blot方法检测肝癌细胞HepG2与肝正常细胞L02中晚期糖基化终末产物受体(RAGE)的表达水平。结果:S100A9在人肝癌组织中的表达较癌旁组织显著增高;GST-S100A9可以促进肝癌细胞HepG2的存活与侵袭,但对肝正常细胞L02无作用;RAGE的表达在HepG2细胞中较在L02细胞中显著升高;RAGE阻断抗体可阻断GST-S100A9对HepG2细胞的促存活与促侵袭作用,表明这些作用是通过RAGE介导的。结论:S100A9促进肝癌细胞HepG2的存活与侵袭依赖于RAGE。     

紫球藻胞外多糖抗柯萨奇B_3病毒活性研究I

要 :采用MTT法研究了紫球藻胞外磺酸化多糖 (ESPS)的细胞毒性 ,在高浓度 (2 5 0 μg/mL)下尚未发现对HEp 2细胞有明显毒性。CPE抑制法发现ESPS能明显抑制细胞的CPE ,在综合、预防、治疗和直接作用四种给药方式下都具有抑制CVB3 的作用。直接作用法的活性最高 ,TI值高达 192 0 6 ,是阳性对照药病毒唑的 31倍。ES PS组分中分子量越大 ,抗病毒活性越高 ,其中分子量大于 30 0k的组分 ,IC50 可低至 0 .75 4 μg/mL ,TI值高达331. 6 ,是病毒唑的 5倍多。结果表明ESPS有强烈的抗CVB3 病毒活性 ,作用机制可能是干扰病毒在细胞表面的吸附及侵染过程。     

细胞自噬与炭疽致死毒素的相互作用

目的：研究炭疽致死毒素在巨噬细胞中引起细胞自噬现象以及细胞自噬对炭疽致死毒素毒性的影响。方法：采用电子显微镜观察、单丹磺酰尸胺（MDC）荧光染色、Western印迹检测研究炭疽致死毒素作用后的巨噬细胞;采用MTT法检测细胞自噬对炭疽致死毒素毒性的影响。结果：采用以上3种方法,在巨噬细胞J774A.1中均可检测到细胞自噬现象;通过诱导或抑制细胞自噬,分别提高或降低了炭疽致死毒素的半数致死浓度。结论：炭疽致死毒素在巨噬细胞内能引起细胞自噬现象;细胞自噬能减弱炭疽致死毒素对巨噬细胞的毒性。     

In Vitro Anti-influenza Virus Activities of Sulfated Polysaccharide Fractions from Gracilaria lemaneiformis

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Synthetic peptide of the sequence 632-642 on myosin subfragment 1 inhibits actomyosin ATPase activity.

A synthetic peptide corresponding to a sequence 632-642 (S632-642) on the myosin subfragment 1 (S-1) heavy chain and spanning the 50/20 kDa junction of S-1 binds to actin in the presence and absence of S-1. The binding of 1.0 mole of peptide per actin causes almost complete inhibition of actomyosin ATPase activity and only partial inhibition of S-1 binding to actin. The binding of S632-642 to the N-terminal segment of actin is supported by competitive carbodiimide cross-linking of S-1 and S632-642 to actin and the catalytic properties of cross-linked acto-S-1 and actin-peptide complexes. These results show that the sequence 632-642 on S-1 is an autonomous binding site for actin and confirm the catalytic importance of its interactions with the N-terminal segment of actin.     

Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6

Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor alpha-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.     

In vitro anti-influenza virus activities of sulfated polysaccharide fractions from <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis>

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Secretion of Carboxypeptidase E from Cultured Astrocytes and from AtT-20 Cells, a Neuroendocrine Cell Line: Implications for Neuropeptide Biosynthesis

Cultured astrocytes have recently been shown to produce certain neuropeptides, as well as neuropeptide processing enzymes. To characterize the secretory pathway in cultured astrocytes, we used the neuropeptide processing enzyme carboxypeptidase E (CPE) as a marker for neuropeptide secretion. Cultured astrocytes and AtT-20 cells, a mouse pituitary-derived neuroendocrine cell line, were labeled with [35S]Met for 15 min and then chased with unlabeled Met. CPE was isolated from either medium or cell extracts using a substrate affinity column. The time course of secretion of radiolabeled CPE was significantly different for cultured astrocytes as compared with AtT-20 cells. CPE was rapidly secreted from the astrocytes after a 30-min lag time, presumably reflecting transport through the endoplasmic reticulum and Golgi apparatus, followed by constitutive secretion. The secretion of radiolabeled CPE was essentially complete by 2 h. In contrast, only a portion of the radiolabeled CPE was secreted from AtT-20 cells over a 2-3-h period, indicating that the majority of newly synthesized CPE is stored, presumably in secretory granules within the AtT-20 cells. The regulation of CPE secretion from astrocytes was also examined. CPE secretion is stimulated two- to threefold by prolonged treatment (3-48 h) with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by treatment with other secretagogues that stimulate CPE secretion from AtT-20 cells (forskolin, isoproterenol, A23187, and vasoactive intestinal peptide) or short (less than 3 h) exposure to TPA. Taken together, these results indicate that the secretory pathway for CPE, and presumably neuropeptides, is substantially different in astrocytes than the secretory pathway for CPE in neuroendocrine cells.     

Chrysin and its phosphate ester inhibit cell proliferation and induce apoptosis in Hela cells

To improve the biological activities of chrysin (CR), we synthesize Diethyl Chysin-7-yl phosphate (CPE: C(19)H(19)O(7)P) and tetraethyl bis-phosphoric ester of chrysin (CP: C(23)H(28)O(10)P(2)) through a simplified Atheron-Todd reaction. The interactions of the CR and CPE with lysozyme were explored by electrospray ionization mass spectrometry (ESI) and fluorescence spectrometry method. Experimental results indicate that CPE could form the noncovalent compound with lysozyme, while the interaction of the CR with lysozyme was not detected. In addition, whether and how the compounds CPE and CP affect proliferation and apoptosis in human cervical cancer Hela cells were investigated. Moreover, the effects of CPE and CP in Hela cells were compared with that of the nonmodified CR compound. The Hela cells were co-cultured with CR, CP, and CPE as experimental groups, respectively, and corresponding control groups treated without CR, CP, and CPE. The proliferation and apoptosis were detected using MTT assay, HCl denatured-methyl green-pyronin staining, PCNA immunohistochemistry and TUNEL techniques. The cell growth IC(50), relative absorbance (RA), proliferating index (PI), PCNA-IR (immunoreactivity IR) integration value (IV), and apoptosis index (AI) were calculated and their correlation was analyzed in each group. The results show that all CR, CP, and CPE could inhibit proliferation and induce apoptosis in Hela cells. Moreover, the effects of CP and CPE were more potent than that of CR. The CP and CPE were proved to be a kind of stronger apoptosis inducers than nonphosphated CR. There was a negative correlation between proliferation and apoptosis. In conclusion, the CR, CP, and CPE could effectively inhibit growth by down-regulated expression of PCNA, and induce apoptosis in Hela cells. The efficiency of the modified CP and CPE preceded nonmodified CR compounds. The CP and CPE may be a new potential anti-cancer drug for therapy of human cervical carcinoma.     

Vascular smooth muscle insulin resistance, but not hypertrophic signaling, is independent of angiotensin II-induced IRS-1 phosphorylation by JNK

Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulin receptor substrate-1 (IRS-1). While the pathway responsible for IRS-1 degradation in this system is unknown, c-Jun NH(2)-terminal kinase (JNK) has been linked with serine phosphorylation of IRS-1 and insulin resistance. We investigated the role of JNK in ANG II-induced IRS-1 phosphorylation, degradation, Akt activation, glucose uptake, and hypertrophic signaling, focusing on three IRS-1 phosphorylation sites: Ser302, Ser307, and Ser632. Maximal IRS-1 phosphorylation on Ser632 occurred at 5 min, on Ser307 at 30 min, and on Ser302 at 60 min. The JNK inhibitor SP600125 reduced ANG II-induced IRS-1 Ser307 phosphorylation (by 80%), IRS-1 Ser302 phosphorylation (by 70%), and IRS-1 Ser632 phosphorylation (by 50%). However, JNK inhibition had no effect on ANG II-mediated IRS-1 degradation, nor did it reverse the ANG II-induced decrease in Akt phosphorylation or glucose uptake. Transfection of VSMCs with mutants S307A, S302A, or S632A of IRS-1 did not block ANG II-mediated IRS-1 degradation. In contrast, JNK inhibition attenuated insulin-induced upregulation of collagen and smooth muscle α-actin in ANG II-pretreated cells. We conclude that phosphorylation of Ser307, Ser302, and Ser632 of IRS-1 is not involved in ANG II-mediated IRS-1 degradation, and that JNK alone does not mediate ANG II-stimulated IRS-1 degradation, but rather is responsible for the hypertrophic effects of insulin on smooth muscle.     

马抗SARS-CoV免疫球蛋白对SARS-CoV GZ-01株中和作用的研究

观察马抗SARS-CoV免疫球蛋白对SARS-CoV GZ-01毒株的中和作用,为马抗SARS-CoV免疫球蛋白用于SARS的治疗提供科学的依据。采用CPE、MTT测定马抗SARS-CoV免疫球蛋白对SARS-CoV GZ-01株的中和作用。结果显示,马抗SARS-CoV免疫球蛋白F(ab’)2治疗性抗体对SARS-CoV GZ-01株具有很好的中和作用,三批马抗SARS-CoV免疫球蛋白的CPE和MTT中和效价分别达到1:6400、1:6400、1:12800,且两种方法测得结果一致。