细胞因子诱导的肿瘤杀伤细胞(CIK)培养条件优化的研究

肿瘤生物治疗目前是继手术、药物、放化疗治疗之后的第四种肿瘤治疗主要手段,目前,临床上应用最广泛的是细胞因子诱导的肿瘤杀伤细胞(CIK).由于CIK培养方式多样,因子选择及配比不同,医疗机构各有不同,造成CIK细胞数量、有效细胞数量、杀伤率有较大的差异.本研究通过对无血清培养基中添加的单克隆抗体CD3,注射用重组人白介素-2(IL-2),采血者自体血浆进行正交实验,得知在无血清培养基中加入CD3 100ng/mL,I-2 700IU/mL,自体血浆2％时,培养效果最好,双阳性有效细胞比率可达97.88％,以效靶比10:1接种,MTT法检测,效应细胞CIK对Hela细胞杀伤率为32.80％.初步观察此种培养条件下的CIK细胞在临床治疗上有一定的疗效.     

DC-CTL体外杀伤人胰腺癌细胞效应研究

研究胰腺癌患者外周血分离培养而来的树突状细胞体外对人胰腺癌细胞BXPC-3、Capan-2的抑制作用.应用各种细胞因子诱导培养从胰腺癌患者外周血分离的单核细胞,获得成熟DC及CIK.胰腺癌组织来源抗原致敏DC,激活T细胞增殖分化为CTL.MTT法检测抗原致敏的DC激活的CTL及单独的CIK对BXPC-3细胞和Capan-2 细胞的杀伤效应.结果显示:胰腺癌抗原致敏的DC,激活肿瘤抗原特异性CTL,体外对BXPC-3和Capan-2胰腺癌细胞产生杀伤作用,分别为75.85%和50.34%;CIK对BXPC-3和Capan-2胰腺癌细胞也有杀伤作用,分别达到62.06%和40.92%,但比抗原致敏的DC激活的CTL的杀伤效应低(P<0.05).胰腺癌患者外周血来源DC在体外能诱导高效的抗胰腺癌细胞免疫反应,提示肿瘤抗原激活的DC作为肿瘤疫苗在胰腺癌的免疫治疗中具有重要价值.     

从脐血单个核细胞定向诱导巨核细胞的生成

采用 SCF、TPO、IL- 3、IL- 6、IL- 1细胞因子 ,从脐血单个核细胞定向诱导巨核细胞的形成。比较了 SCF+ TPO+ IL- 1、SCF+ TPO+ IL- 3、SCF+ TPO+ IL- 63种细胞因子组合对刺激巨核细胞生成的作用 ,经体外培养 8d后 ,CD41 + 细胞占培养物的比例分别达到 ( 5 .64± 0 .77) %、( 5 .73± 1 .2 4 ) %和 ( 2 0 .1± 2 .5 3) % ;每 1 0 4个细胞可形成巨核祖细胞集落形成单位 ( CFU- MK)为( 1 3.7± 5 .7)个、( 1 5 .0± 3.6)个和 ( 93.7± 1 6.0 )个。在 SCF+ TPO+ IL- 6体系中增加 IL- 6的浓度 ,可提高培养液中 CD41 + 细胞的纯度。当 IL- 6的浓度从 1 0μg/L增加到 5 0μg/L时 ,CD41 + 细胞的比例可从 ( 2 0 .1± 2 .5 3) %提高到 ( 2 6.81± 3.2 0 ) % ,但每 1 0 4个细胞形成 CFU- MK数目却从( 93.7± 1 6.0 )个减少到 ( 4 5± 8.6)个 ;这说明 IL- 6对巨核细胞的刺激主要作用在其发育的后期。采用 SCF+ TPO+ IL- 3+ IL - 6细胞因子组合 ,由 1× 1 0 6个单个核细胞培养一周后可诱导出 ( 1 .61±0 .5 8)× 1 0 5个 CD41 + 细胞 ,占培养物的比例可达到 ( 1 8.8± 1 .64) % ,每 1 0 4个细胞形成 CFU- MK数目可达到 ( 1 2 1 .8± 1 0 .3)个。这一结果为进一步体外大量扩增巨核细胞提供了基础     

大鸨的随机扩增多态DNA分析与种内亲缘关系研究

利用随机扩增多态DNA技术对大鸨(Otis tarda)6个个体进行了随机扩增多态DNA分析,共筛选出有效随机引物14条,利用所得的随机引物对每只大鸨的基因组DNA进行扩增,共得到327个扩增片段,其中共有片段150个,根据聚类分析所得到的树状图确定了6只大鸨的亲缘关系.     

一种人外周血NK细胞高效扩增的方法

目的 探索从人PBMC中高效扩增CD3~- CD56~+NK细胞的方法。方法 使用干细胞生长培养基,在抗CD3单抗和IL-2的作用下,从健康成人PBMC中诱导扩增CD3~- CD56~+NK细胞,并用流式细胞仪检测扩增细胞的免疫表型。结果 PBMC经抗CD3单抗、IL-2联合作用获得大量增殖,在14d时扩增54.3±5.5倍,含有(52.4±7.9)％CD3~- CD56~+NK细胞。结论 可使用干细胞生长培养基,在抗CD3单抗和IL-2协同刺激下大量扩增NK细胞,为应用NK细胞进行肿瘤过继免疫治疗提供了一种简单有效的扩增NK细胞的方法。     

(R)-Roscovitine诱导Hela细胞凋亡的分子机制

以Hela细胞为模型,探讨细胞周期蛋白依赖性激酶抑制剂(R)-roscovitine对Hela细胞凋亡诱导的可能途径,进一步了解其诱导肿瘤细胞凋亡的分子机制.结果显示:(R)-roscovitine明显抑制Hela细胞的增殖,在17.5～140.0μmol.L-1浓度作用12,24,48h后,Hela细胞的增殖抑制率分别为8.48%～33.76%,25.19%～86.41%和47.90%～88.08%;典型凋亡的DNA ladder被诱导,且凋亡率随药物浓度的增加而升高;经半胱天冬酶-3(caspase-3)特异性抑制剂Ac-DEVD-CHO或半胱天冬酶泛抑制剂Z-VAD-FMK处理后,(R)-roscovitine对Hela细胞凋亡的诱导被抑制约20%,说明半胱天冬酶依赖的途径参与了(R)-roscovitine对Hela细胞凋亡的诱导;(R)-roscovitine诱导凋亡诱导因子(AIF)从线粒体释放细胞核中,且Ac-DEVD-CHO和Z-VAD-FMK没有抑制AIF的释放,这意味着(R)-roscovitine能够诱导Hela细胞凋亡,其机制是通过caspase与非caspase依赖两种途径.     

新型邻菲啰啉钌配合物的合成、聚集诱导发光性质及细胞成像

以邻菲啰啉为母体,设计合成了一种具有聚集诱导荧光增强(AIE)性质的钌配合物1,并利用核磁共振氢谱、质谱和红外对其结构进行了表征.通过紫外-可见吸收光谱、荧光光谱、细胞毒性及细胞成像等实验研究了配合物1的光学性质及其在细胞内的应用.结果表明：配合物1的荧光发射峰在600 nm,且具有明显的AIE性质,当水含量为80%时,其荧光强度增强约5.2倍;此外,配合物1的生物毒性较小,可应用于固定细胞中的细胞成像.     

NO诱导血管平滑肌细胞凋亡中Ca~(2 )的作用

探讨了ＮＯ诱导血管平滑肌细胞凋亡与细胞内游离Ｃａ２＋之间的关系．通过粘附式细胞仪和Ｃａ２＋ 荧光探针Ｆｌｕｏ－３／ＡＭ,检测分析了ＮＯ在供体ＳＮＡＰ的作用下,血管平滑肌细胞中游离Ｃａ２＋浓度的变化; 又通过ＳＮＡＰ与维拉帕米、ＥＧＴＡ、肝素钠、普鲁卡因共同孵育的方法,观测了Ｃａ２＋浓度变化在细胞凋亡中 的作用．得出ＳＮＡＰ能使细胞中游离Ｃａ２＋浓度升高,而胞外Ｃａ２＋内流在其中起主要作用;并且阻断胞外 Ｃａ２＋内流能够抑制ＳＮＡＰ所诱导的血管平滑肌细胞的凋亡．提示了胞内Ｃａ２＋浓度升高可能是ＳＮＡＰ诱导血管 平滑肌细胞凋亡的一条途径．     

处理酿酒厂和乙酸废水生物颗粒中的细胞外聚合物

处理酿酒厂废水的生物颗粒与乙酸的生物颗粒相比,具有相当强的抗损耗和溶解能力是因为较高含量的ECP.每mg由Methanothrix细菌组成的后者平均只含有0.09 mg的ECP.但是前者含有0.15 mg的ECP,主要是因为高含量的ECP存在于位于生物颗粒表面的hydrolytic/fermentative 细菌和中间层的syntrophic细菌之中的缘故.     

一种采用微波炉加热快速提取细菌DNA用于PCR扩增的方法

利用微波加热原理,探索一种快速、简单、高效提取细菌DNA用于常规PCR快速检测的方法.在微波炉额定功率800 W条件下分别对大肠杆菌、金黄色葡萄球菌、单增李斯特氏菌、副溶血弧菌、沙门氏菌以及志贺氏菌6种常见食源性病原菌进行微波处理,离心取上清,获取细菌DNA,将样本DNA进行PCR扩增及琼脂糖凝胶电泳进行验证.并进一步采用微波加热方法针对6种病原菌最低检出浓度和混合提取6种病原菌DNA的PCR检测进行分析.6种病原菌在微波炉中加热40-130 s的上清DNA样本均能用于常规PCR扩增.针对检出浓度的分析表明,副溶血弧菌的最低检出浓度为103cfu/ml,金黄色葡萄球菌为104cfu/ml,肠出血性大肠杆菌为105cfu/ml,肠炎沙门氏菌和福氏志贺氏菌的最低检出浓度为106cfu/ml,针对单核细胞增生李斯特氏杆菌的的最低检出浓度为107cfu/ml.进行6种混合菌微波共提取时,其中5种病原菌的特异性基因都能扩出,并且条带很亮.该微波提取方法具有快速,简单,高效的特点,能满足大部分食源性病原菌的常规PCR检测需求,大大节约了时间和成本,具有广泛的适用性,为细菌快速分子检测提供了简便手段.     

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

Critical role of Toll-like receptor signaling in NK cell activation

Toll-like receptors (TLRs) and NK cell receptors are the most important receptor superfamilies in innate immunity. TLRs act as the sensor of external pathogens, while NK cells detect alterations in endogenous protein expression on target cells through activating and inhibitory receptors. Accumulating data has demonstrated that TLRs and NK cell receptors can coordinate and regulate each other during immune responses, which contributes to the initiation of innate response and the priming of adaptive responses. TLRs can activate NK cell function directly or with the help of accessory cells in a cytokine or cell-to-cell contact dependent manner. More understanding of the recognition of innate receptors and interactions between them may provide important insights into the design of effective strategies to combat tumor and microbial infections. In this review, we summarize how TLRs and NK cells discriminate the self or non-self components respectively. And importantly, we pay more attention to the role of TLR sig-naling in induction of NK cell activation, responses and the crosstalk between them.     

I(L)型诱导空间的分离性

证明了(L^X,δ)是次T0，T0(T’0)，Tl(T'1)，T2，正则(T3)，正规(T4)，STl，ST2，ST3，ST4和完全正则(T3 1/2)空间当且仅当(I(L)X，ω(δ)是次T0，T0(T’0)，Tl(T’1)，T2，正则(T3)，正规(T4)，ST1，ST2，ST3，ST4和完全正则(T 3 1/2)空间。同时给出(I(L)^X，ω(δ))是包含式正则、正规空间蕴含(L^X，δ)是包含式正则、正规空间。     

基于灰色理论的多参数海洋立管涡激振动试验研究

通过模型试验的方法对多参数作用下的海洋立管的涡激振动进行了研究。试验参数包括:立管模型材料、顶张力、边界条件以及流速。模型材料选取了铝(Al)、有机玻璃(PMMA)和硬质塑料(UPVC)三种;流速采用0.2 m/s、0.6 m/s和1 m/s;边界条件采用两端铰接(S-S)和铰接-固接(S-F)两种,顶张力选取为5 N和65 N。通过电测法获得动态应变数据,得到立管的顺流向振动响应、横流向振动响应以及漩涡脱落频率;利用灰色理论分析不同参数对立管涡激振动响应的影响。结果表明,立管材料对立管的振动起着主要影响,各参数对漩涡脱落频率的关联序为r_(01)r_(02)r_(03)r_(04)(r_(01)为材料,r_(02)为流速,r_(03)为边界条件,r04为顶张力);即立管材料、流速与边界条件对漩涡脱落频率影响大,顶张力较其他三个因素影响稍弱。     

Induced pluripotent stem cell (iPS) technology: promises and challenges

In 2006, an article published in Cell by Shinya Yamanaka took by surprise the stem cell research community. By performing systematic retroviral transduction of factors enriched in embryonic stem (ES) cells, the authors demonstrated the reprogramming of mouse fibroblasts into an ES cell-like state. These cells, baptized iPS (induced pluripotent stem) cells, were immediately recognized as a ground-breaking discovery. Subsequently, the same authors and other groups reported a similar achievement with human fibroblasts. Two years later, the number of top quality papers on iPS is astonishing, and interest in the scientific community has risen to a fever pitch. But although iPS has the potential to revolutionize Regenerative Medicine, important questions still remain unanswered. Work from multiple laboratories worldwide including ours is focused on deciphering the molecular mechanisms of iPS, and trying to improve the technique to make it suitable for the clinic. In this review article we briefly discuss the past, present and future of iPS, with emphasis on urgent issues to be solved. Supported by the National Nature Science Foundation of China (Grant Nos. 30725012, 30630039 and 90813033), Knowledge Innovation Project of Chinese Academy of Sciences (Grant No. KSCX2-YW-R-48), National Key Basic Research and Development Program of China (Grant Nos. 2006CB701504, 2006CB943600, 2007CB948002, 2007CB947804. 2007CB947900) and Guangzhou Science and Technology Development Funds (Grant No. 2008A1-E4011)     

Sources of cumulus expansion enabling factor (CEEF) in porcine follicles

It was shown that expansion of porcine cumulus did not depend on oocyte-secreted factor(s), and it is therefore presumed that porcine CEEF may not be produced exclusively by the oocyte. In this experiment, we used mouse oocytectomized complexes (OOX), which were incapable of CEEF production, to assess the secretion of CEEF by evacuated zona, oocytes of different quality and somatic cells in the porcine follicles. The results showed that: (ⅰ) Evacuated zonae from both porcine and mouse oocytes did not produce CEEF. (ⅱ) Porcine oocytes of A, B and C types from 3—6 mm follicles were not significantly different in both production and activity of CEEF. (ⅲ) Both porcine OOX from 3—6 mm follicles and granulose cells from < 1 mm follicles secreted CEEF in a large quantity, independent of gonadotropins; mural granulose cells from 3—6 mm follicles, however, produced neglectable amount of CEEF. (ⅳ) The follicular fluid from 3—6 mm porcine follicles contained CEEF activity that was concentration-dependent, and thus it enabled cumulus expansion in 60% mouse OOX when used at 10% of concentration, but the expansion rate of mouse OOX decreased to 9% when the concentration was increased to 50%. (ⅴ) Mouse OOX cultured in porcine CEEF-containing M199 expanded only in the presence of gonadotropins, suggesting that the activity of porcine CEEF is hormone-dependent.     

幽门螺杆菌培养滤波对IL-8及胃粘膜增殖细胞核抗原表达的影响

目的:研究幽门螺杆菌(Helicobacter pylori,Hp)代谢产物对炎性细胞因子白细胞介素8(interleukin-8,IL-8)表达的影响以及在胃粘膜上皮细胞增殖中的作用。方法:用CagA阳性Hp培养滤液给大鼠灌胃,2周后,采用ELISA法检测鼠血清中的IL-8水平,免疫组织化学SP(streptavidin/perosidase)法检测胃粘膜增殖细胞核抗原(proliferation cell nuclear antigen,PCNA)的表达。结果:灌服Hp滤液组与对照组比较,血清IL-8升高,胃粘膜PCNA标记指数阳性率升高,均有显著性意义。结论:CagA阳性Hp培养滤液可促进炎性因子IL-8的表达,促进胃粘膜上皮细胞的增殖,可能促进胃癌的发生。     

草地贪夜蛾(Spodoptera frugiperda)细胞在生物反应器中培养条件初探

对草地贪夜蛾（ Ｓｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａ） Ｓｆ ９ 细胞在２ Ｌ Ｂｉｏｓｔａｔ Ｒ Ｍ Ｄ 型搅拌式生物反应器中进行了培养,并对细胞的生长状况、葡萄糖的消耗等进行了测定;对重组病毒的增殖、外源基因的表达进行了初步探索．结果表明：细胞在该生物反应器中生长良好,细胞群体倍增时间３３．４ ｈ．病毒感染率大约在 ８９．８％ 以上,并在细胞中成功地表达了半乳糖苷酶基因     

Innate immune recognition and regulation in liver injury: A brief report from a series of studies

The discovery of innate immune receptors and the emergence of liver immunology （high content of NK and NKT cells in liver） led to the second research summit in innate immunity since the finding of NK cells in the middle 1970s. Liver disease is one of the most dangerous threats to humans, and the progress in innate immunology and liver immunology made it possible to re-explain the cellular and mo- lecular immune mechanisms of liver disease. In the past ten years, we have found that innate recognition of hepatic NK and NKT subsets were involved in murine liver injury. We established a novel NK cell-dependent acute murine hepatitis model by activating Toll-like receptor-3 （TLR-3） with an injection of poly I：C, which may mimic mild viral hepatitis （such as Chronic Hepatitis B）. We observed that a network of innate immune cells including NK, NKT and Kupffer cells is involved in liver immune injury in our established NK cell-dependent murine,model. We noted that TLR-3 on Kupffer cells activated by pretreatment with poly I： C might protect against bacterial toxin （LPS）-induced fulminant hepatitis by down-regulating TLR-4 function, while TLR-3 pre-activation of NK cells might reduce Con A-induced NKT cell-mediated fulminant hepatitis by blocking NKT cell recruitment to the liver. We also found that the oversensitivity to injury by immune stimulation in HBV （hepatitis B virus） transgenic mice （full HBV gene-tg or HBs-tg） correlated to the over-expression of Real, an NKG2D （natural killer cell group 2D） ligand of NK cells or CDld, a ligand of TCR-V14 of NKT cells, on HBV＋ hepatocytes, which leads to an innate immune response against hepatocytes and is critical in liver immune injury and regeneration.     

I(L)诱导Fuzzy拓扑空间的完全正则性

证明完全正则性是Ｉ（Ｌ）好的推广,即诱导空间（Ｉ（Ｌ）Ｘ,ω（δ））是完全正则空间当且仅当（ＬＸ,δ）是完全正则空间．