体外培养条件下不同氧浓度对几种细胞生长的影响

观察多级氧浓度对体外培养条件下小鼠原代骨髓基质细胞、小鼠3T3-L1前脂细胞及大鼠BRL肝细胞生长的影响,探究不同类型细胞体外增殖的最佳O2体积分数以及对低氧和高氧耐受力的差异.实验结果显示,在10%~18%O2范围内骨髓基质细胞增殖最快、活力最强,而3T3-L1前脂细胞及BRL肝细胞生长的最适O2体积分数为5%.O2浓度过低严重损害这几种细胞在培养下的生存能力,O2浓度过高有致死性毒性作用.这些结果表明,原代与长期传代细胞最佳生长的适宜氧浓度有一定的差异,对低氧和高氧的耐受力也强弱不同.     

GNL3对脑胶质瘤干细胞的调控研究

肿瘤干细胞是一小群特异的肿瘤细胞,被认为是肿瘤发生,耐药和复发的主要原因。GNL3(Guanine nucleotide-binding protein-like 3) 是MMR1/HSR1 GTP结合蛋白家族成员,在干细胞增殖中发挥重要作用。本实验室通过质谱筛选,发现GNL3蛋白在脑胶质瘤干细胞(Glioma stem cell,GSC)中特异性高表达。目前关于GNL3在脑胶质瘤干细胞中的功能及其作用机制未见文献报道。为了研究GNL3在GSC中的功能,我们利用慢病毒感染细胞体系,在GSC中应用3个不同的靶序列对GNL3基因的表达进行稳定干涉;利用Western Blot对GNL3的干涉效果进行检测。实验结果表明,在稳定敲低GNL3表达的GSC中,胶质瘤干细胞的细胞活力和肿瘤细胞球(Tumor sphere)的形成能力明显受到抑制,表明GNL3蛋白对GSC的增殖能力及自我更新能力具有重要调控,提示GNL3在胶质母细胞瘤的肿瘤发生过程中发挥一定作用。     

人类心房肌细胞的原代培养与鉴定

探索人心房肌细胞的原代培养方法。取心外科手术患者(5/12~57岁,平均6.5岁)常规切除的右心耳,利用酶消化法原代培养心房肌细胞并进行光镜和免疫细胞化学鉴定。结果急性分离的细胞成圆形,1 d后开始贴壁。贴壁的心肌细胞部分聚集成团,成梭形或三角形。随着培养时间延长,细胞逐渐向四周伸展。免疫细胞化学分析显示90%以上的细胞呈横纹肌肌动蛋白染色阳性。说明利用酶消化法成功培养出人心房肌细胞,为深入研究人心房肌细胞基因与功能打下了基础。     

多级色谱纯化牛胎盘免疫调节多肽及性质测定

使用DEAE Sepharose CL-6B阴离子交换色谱、Sephadex G-25 凝胶色谱和Sephasil C18反相高效液相色谱等分离纯化技术,采用体外培养淋巴细胞增殖试验,从牛胎盘提取液中分离出了具有免疫调节活性的多肽。免疫调节多肽对淋巴细胞增殖的促进作用具有剂量依赖性。纯化后的免疫调节多肽经过毛细管电泳(CE)鉴定为单一组分。毛细管等电聚焦电泳(CIEFE)测定多肽的等电点是3.82。基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS)测定多肽的相对分子质量为2134。蛋白质测序仪测定多肽的序列为Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr。     

Maturation of Osteoblast-Like Saos-2 on Hydroxyapatite Ceramics

Hydroxyapatite ceramics (.HA) has been proved to be excellent in biocompatibility and bioactivity.However, limited information is available concerning how HA ceramics affects the maturation of es-tcoblasts in molecular biological level /n v/tro. This study examines the mRNA expression and protein production of hone-related genes in estcoblast-like cell line(Saos-2) cultured on HA disks. Saos-2 cells are seeded onto the substrates and cultured for 18 days. Harvested cells are tested for the cell growth rate, expression of mRNAs for esteocalcin and alkaline phosphatase, and protein production of bone sialo-protein and esteocalcin. MTS assay shows that cell proliferates well on HA ceramic substrate. After 9d,bone sialoprotein and esteocalcin protein production in SAPS-2 increases more on HA surfaces than on control material. As bone sialoprotein and esteocalcin are the genes to be highly expressed at the late stage of estcoblast differentiation, this study reveals that after long time culture in HA, HA can induce Saos-2 maturation. The behavior of Saos-2 on HA surfaces revealed in this study provides valuable infor-mation for the understanding of the biocompatibility and bioactivity of HA ceramics.     

秋石斛兰离体快速繁殖研究

以秋石斛兰小苗的茎尖为材料,进行离体繁殖研究,结果表明:原球茎诱导以NX+6-BA1.5 mg·L~(-1)+NAA 0.15 mg·L~(-1)培养基较好,诱导率达57.7％;原球茎块在NX+6-BA 1.5 mg·L~(-1)+NAA 0.2 mg·L~(-1)培养基上增殖效果好,35 d增殖达7.52倍,接入的原球茎块体积大,增殖倍数高,在接种后20～30 d是原球茎增殖最快的时期;原球茎在MS+6-BA 3,0 mg·L~(-1)培养基上出芽多,50 d可分化出38.1个小芽,接种后40～50 d,分化出芽数增加最快;生根培养以1/2 MS+IBA 0.4 mg·L~(-1)最好,生根率达98.7％。     

Aurora-A对2型糖尿病小鼠模型中胰岛细胞增殖的影响

为研究Aurora-A在2型糖尿病小鼠中对胰岛细胞增殖的影响,通过Western blot、免疫组织化学染色、增殖细胞核抗原(proliferating cell nucler antigen,PCNA)染色等方法研究了Aurora-A在2型糖尿病小鼠胰腺组织中的表达以及抑制AuroraA后糖尿病小鼠胰岛细胞的增殖情况。将32只雄性C57BL/6J小鼠随机分为4组,每组8只,分别为空白组、高脂饮食组、模型组和治疗组。模型组和治疗组采用高脂饲料(high fat diet,HFD)联合链脲佐霉素(Streptozotocin,STZ)腹腔注射造模,造模成功后治疗组以Aurora-A抑制剂连续灌胃2周;模型组则以等量的生理盐水灌胃。经药物干预后采用PCNA染色检测各组小鼠胰岛细胞的增殖情况。结果表明:与空白组相比,Aurora-A在模型组胰腺组织中明显表达升高;与模型组相比,治疗组小鼠经Aurora-A抑制剂治疗后胰岛细胞增殖率明显降低。可见,Aurora-A在2型糖尿病小鼠胰腺组织中表达升高;并对胰岛细胞的增殖起着重要的作用。     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Peptides and epithelial growth regulation

Summary There is now considerable evidence implicating several peptides in the control of gastrointestinal epithelial cell proliferation and cell renewal. While some of these may act directly, many may be involved in regulating the powerful trophic effects of the intake and digestion of foold on the gut epithelium.—Several peptides have been associated with the regulation of intestinal cell proliferation. There is little doubt that gastrin is trophic to the stomach, but, its role in the rest of the gastrointestinal tract is debatable. Enteroglucagon has often been associated with increased intestinal epithelial proliferation, but at the moment all the evidence for this is circumstantial. The effects of peptide YY and bombesin warrant further study. The availability of recombinant epidermal growth factor (EGF) has recently enabled us to demonstrate a powerful trophic response to infused EGF throughout the gastrointestinal tract. The increasing availability of peptides will eventually allow the rigorous in vivo evaluation of the trophic role of these potentially very important peptides.     

血清hK2 ELISA方法的建立及在前列腺癌诊断中的应用

目的通过建立血清hK2 ELISA检测方法,探讨血清hK2水平对前列腺癌的诊断意义,为临床鉴别前列腺癌与前列腺增生提供一种特异、敏感、实用的方法.方法利用hK2单克隆抗体建立检测血清hK2水平的ELISA方法,并利用本实验建立的方法对30例前列腺癌患者,26例前列腺增生患者,30例正常对照的血清进行hK2水平的检测.结果 1)血清hK2水平测定值(A450nm)分别为:a.前列腺癌组(0.701±0.113);b.前列腺增生组(0.212±0.145);c.正常对照组(0.198±0.015).前列腺癌组血清hK2水平与前列腺增生、正常对照组相比较有显著差异(P<0.01).2)本研究建立的检测血清hK2水平的ELISA方法的敏感度为83.3%,特异度为94.6%,准确度为87.2%.结论 1)前列腺癌患者血清hK2水平与前列腺增生患者及正常人血清hK2水平差异显著,不存在重叠.血清hK2水平能够对前列腺癌、前列腺增生进行鉴别诊断.2)在鉴别前列腺癌和前列腺增生方面,本研究建立的检测血清hK2水平的ELISA方法,与临床上应用的病理学诊断方法符合率高,具有临床实用价值.