Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Melanoma cell growth inhibition by beta gamma-CAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin

In vertebrates, non-lens beta gamma-crystallins are widely expressed in various tissues but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

抗黑色素瘤ScFv-超抗原SEA重组免疫毒素的构建与表达

提取鼠抗人黑色素瘤杂交瘤细胞株HB8759的总RNA，反转录成cDNA，用抗体可变区混合引物扩增出全套重、轻链可变区基因（VH、VLDNA），通过（Gly4Ser）3连接肤基因把VH和VL基因装配成单链抗体（ScFv）基因，将其克隆到噬菌粒载体pCANTABSE中，构建单链抗体噬菌体抗体库。用LIBr黑色素瘤细胞对抗体库进行了3轮亲合筛选。随机挑选克隆进行hage-ELISA鉴定，结果获得了2株具有较高ELISA活性的噬菌体单链抗体。序列测定证实得到的ScFv符合抗体可变区的结构特点，与已发表的鼠抗体可变区基因有较高的同源性。采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肤的金黄色葡萄球菌肠毒素A（SEA）基因进行融合，并将融合基因克隆于pET28-a表达载体的H招标签下游。SDS-PAGE分析表明，两种构建方法均表达了相对分子量约SOkD的蛋白条带，与预期目的蛋白分子量相符，主要以包涵体的形式存在，表达量占菌体蛋白的27％。将重组质粒在大肠杆菌中诱导表达，用盐酸肌溶解包涵体，Ni-NTA鳌合层析柱一步法纯化包涵体，再通过透析使目的蛋白复性。凝胶灰度扫描显示蛋白纯度达90％，凝胶电泳呈单一条带，蛋白量达0.47mg／mL。用健康人外周血单个核细胞作为效应细胞，LDH法检测ScFv-SEA融合蛋白对LiBr黑色素瘤细胞、MCF7乳腺癌的体外抑制率。结果表明该融合蛋白可以通过活化效应细胞，对表达相关抗原的肿瘤细胞产生有效的抗增殖作用，而对不表达该抗原的肿瘤细胞作用不显著。说明该融合蛋白赋予了SEA抗黑色素瘤特异性。以上实验结果为我们研究SEA对黑色素瘤的靶向杀伤作用奠定了可靠的实验基础。

Studies on advantages of heavy ions in radiotherapy compared with gamma-rays

Hepatoma and melanoma cells were exposed to C-12(6+) beams generated by HIRFL facility and gamma-rays and the cell response was studied by colony assays as well as the analysis of RBE of carbon ions was evolved. The survival curves of cells irradiated by heavy ions were different from those of cells irradiated by gamma-rays. And two kinds of cell showed the obvious discrepancy in response to the photon and ion irradiation. The results showed that heavy ions have special physical properties and mighty potency to kill cell in both single and fractional irradiation meanwhile it can kill tumor cells with high radioresistance more efficiently. When involved in clinical therapy, heavy ions will enhance the therapy efficiency and decrease the suffering of patients because it can impair the repair for sublethal damage of cells which can lead to fewer irradiation fractions.

Early effects of low dose C-12(6+) ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose C-12(6+) ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy C-12(6+) ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supematant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-gamma and TNF-alpha in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy C-12(6+) ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) C-12(6+) radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDL (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

辐射联合p53基因转导对人黑色素瘤细胞系生物学效应的研究

恶性黑色素瘤具有早期高度转移潜能，尽管常常检测到野生型p53基因，却对常规放化疗高度抗拒，因此，寻求有效治疗黑色素瘤的方法十分重要。本文就如何将放射治疗与基因治疗有机结合、外源性p53对不同p53状态的黑色素瘤细胞生长抑制和辐射增敏作用，以及外源性P53蛋白对不同传能线密度（LET）射线辐照诱导肿瘤细胞死亡途径的影响做了一些粗浅研究。 首先，采用复制缺陷的重组腺病毒载体(AdCMV-p53)介导人野生型p53基因转染1 Gy X-射线预辐照的黑色素瘤细胞系A375(wt p53)和WM983a(mu p53)，RT-PCR检测mRNA水平，流式细胞仪测定细胞周期阻滞及外源性P53蛋白表达情况,克隆形成率测定辐射后细胞存活率。用携带报道基因的复制缺陷重组腺病毒载体AdCMV-GFP（绿色荧光蛋白）作为对照。结果显示1 Gy X-射线辐照可明显增加AdCMV-p53对A375和WM983a细胞系的基因转导效率，转导的外源性野生型P53可在两种细胞中高效表达，并诱导细胞周期G1期阻滞；单纯转导p53对A375(wt p53)细胞无明显诱导凋亡和生长抑制效应，但可部分诱导WM983a(mu p53)细胞凋亡；而转导p53基因48小时后给予X-射线辐射，两种细胞克隆存活率较其对照组均明显减低；外源性p53基因对WM983a(mu p53)细胞的辐射增敏作用较A375(wt p53)细胞明显。这些结果表明小剂量辐射既可有效增加腺病毒介导的p53转导，又不会对患者产生明显副作用。而且，外源性野生型p53可明显增加黑色素瘤细胞系A375(wt p53)和WM983a(mu p53)的辐射敏感性。提示p53是基因治疗黑色素瘤较好的侯选基因，也为临床上放疗联合基因治疗恶性黑色素瘤提供了实验室依据。 其次，观察了外源性P53蛋白对不同传能线密度（LET）射线辐照诱导肿瘤细胞凋亡和坏死的影响，并探讨其可能的机理。人黑色素瘤细胞系A375（wild-type p53）经携带人野生型p53基因的腺病毒载体（AdCMV-p53）感染后分别给予X-射线和碳离子束照射，采用克隆形成法测定细胞辐射敏感性，Hoechst33258和吖啶橙-溴化乙锭双染，荧光显微镜下观察细胞凋亡和死亡。结果发现：（1）高LET射线辐照时，A375细胞和转导人野生型p53基因的A375细胞（A375/p53）的辐射敏感性没有明显差异；（2）虽然辐射诱导细胞凋亡比例的增加依赖于LET，但是无论高LET或低LET，外源性P53蛋白均可有效诱导细胞凋亡。（3）高LET射线辐照时，A375细胞的坏死细胞明显高于A375/p53细胞。提示尽管高LET射线辐射对A375和A375/p53细胞的存活无明显影响，但是对细胞凋亡的诱导却部分依赖于P53蛋白的功能，提示P53蛋白可能在调节细胞死亡类型中发挥重要作用。这对临床应用高LET射线辐射联合p53基因治疗恶性黑色素瘤有一定参考意义

p53基因转导联合重离子辐射引起小鼠黑色素瘤细胞生长抑制及其机制的探讨

为了将放射治疗与基因治疗有机结合起来以寻求有效治疗恶性黑色素瘤的方法，采用了AdCMV-p53（AdCMV-GFP）转染B16细胞联合重离子（或X－射线）辐照的方法，观察辐射对基因转移效率的影响、外源性P53蛋白对重离子辐照诱导肿瘤细胞生长抑制和辐射增敏作用，以及外源性P53蛋白对重离子辐照诱导肿瘤细胞内蛋白表达的变化，现将本工作结果总结如下： 1.重离子照射可增加腺病毒载体介导p53基因转导效率，而且先转染后辐照法比先辐照后转染法能更显著的地增加基因转导效率。这样在最大限度提高基因转导效率的基础之上，同时又可以减少病毒使用量及辐照剂量。 2.p53基因转导联合重离子辐照能明显抑制细胞生长，诱导细胞凋亡，促进G0/G1期细胞阻滞。说明外源性野生型p53基因导入联合辐照可增加黑色素瘤细胞系B16的辐射敏感性。 3.重离子照射比X-射线照射能更明显增加腺病毒载体介导p53基因转导效率和G0/G1期细胞所占比例，可能是由于两种射线能量沉积的方式不同造成的。 4.重离子辐照联合p53基因转导诱导B16细胞中细胞信号通路发生变化，使得P53和P21表达明显增多，同时MDM2表达随时间而减少。推测导入的p53基因联合重离子辐照改变细胞内信号通路，从而诱导细胞凋亡和细胞周期阻滞