膜结合型干细胞因子促进白血病细胞K562的增殖和集落形成

目的:探讨膜结合型干细胞因子(membrane-bound stem cell factor,m-SCF)在白血病细胞中的作用。方法:克隆并构建携可溶型干细胞因子(soluble stem cell factor,s-SCF)前体的真核表达质粒pTARGET-s,采用Overlap PCR法去除外显子6序列,进一步构建m-SCF的表达质粒pTARGET-m,DNA测序鉴定。通过脂质体介导分别将上述载体和空载体pTARGET转染白血病K562细胞,用G418筛选稳定表达细胞株,并用RT-PCR、Western blotting法鉴定。通过CCK-8细胞增殖实验以及集落形成实验观察不同细胞株体外增殖特点。结果:成功构建了s-SCF和m-SCF的真核表达载体,获得了稳定转染细胞株K562-V、K562-S、K562-M。U底培养条件下,K562-M增殖能力明显高于K562-V和K562-S(均P<0.01)。K562-M集落形成率显著高于K562-V和K562-S(均P<0.01),且集落形态大于其他两种细胞。结论:m-SCF与C-kit之间的并置性作用显著促进白血病细胞的增殖。

阳和汤对乳腺癌干细胞CD90、IL-8mRNA表达水平的影响

目的:探讨阳和汤含药血清对乳腺癌干细胞HMLER90^(hi)增殖的影响及其作用机制。方法:将20只雌性SD大鼠随机分为阳和汤低、中、高剂量组和空白对照组,按低、中、高剂量分别灌胃阳和汤,空白对照组给予生理盐水,连续给药3天,制备阳和汤含药血清和空白血清。MTT法检测各组在含药血清干预24小时、48小时、72小时后对HMLER90^(hi)细胞增殖的影响;qRT-PCR检测各组细胞CD90、IL-8mRNA表达情况。结果:阳和汤含药血清干预24小时、48小时、72小时后,与空白对照组相比,阳和汤各剂量含药血清组细胞增殖的抑制率均增高,且对细胞增殖的抑制作用呈时间与剂量依赖性,差异具有统计学意义(P<0.05)。阳和汤各剂量含药血清组CD90、IL-8mRNA表达量均降低,且呈时间与剂量依赖性,差异具有统计学意义(P<0.05)。结论:阳和汤可抑制乳腺癌干细胞HMLER90^(hi)的增殖,其作用机制可能与其抑制单核巨噬细胞近分泌通路的传导有关。

Both Juxtacrine and Paracrine Signaling Indispensable in Spermatogonial Stem Cell Cultures

Objective To prove that juxtacrine and paracrine signaling are essential in the culture of spermatogonial stem cells （SSCs） with Sertoli cell feeder layer in vitro. Methods Mice aged 7 d were chosen to harvest testes. A two-step enzyme digestion method was applied in testis suspension. The SSCs and Sertoli cells were separated by adherence distinguishing methods and biologically identified by immunofluorescence and Oil Red 0 staining methods. Flow cytometry was used to analyze purity of SSCs. Three groups were constructed according to different culture conditions. SSC and Sertoli cell co-culture group, SSC conditional culture group and SSC routine culture group. The conditional medium was collected from supernate of culture Sertoli cell in vitro and double-concentrated with DMEM/F12 and fetal bovine serum in a proportion of 4.5 ： 4.5 ： 1. The routine medium was DMEM/F12 containing 10% fetal bovine serum. Adherence rates were measured by Trypan blue staining. Absorbance of SSCs of each group was measured by MTT assay and proliferation curves shown to demonstrate proliferative features of SSCs. Proliferative features and colony formation were observed by inverted microscope. With 24 h difference in adherence rates, proliferations were compared and analyzed.Results The adherence rate of co-culture group was greater than that in the others（P〈0.05）, with insignificant difference in conditional culture group and routine group （P〉0. 05）. SSCs of co-culture group showed stable proliferation immediately following inoculation..4 stable colony formed within 7-10 d and maintained for 30 d. SSCs in conditional culture group and routine group decreased rapidly following transient proliferation. Conclusion The actions of SSCs in Sertoli cell cultures in vitro depended on both juxtacrine and paracrine signaling, Sertoli cell paraerine signaling was unable to promote SSC adherence and proliferation alone.

Notch-1在人胶质瘤细胞系U251细胞中的邻分泌机制探讨

目的探讨Notch-1在人胶质瘤细胞系U251细胞中的邻分泌机制。方法用免疫荧光组织化学方法检测单个孤一和聚集相邻的两个U251细胞的Notch-1及配体delta-like-1的表达水平。封闭U251细胞的Notch-1受体,采用SYBR荧光实时定量PCR法检测Notch-1配体delta-like-1 mRNA在不同封闭时间组的表达水平。结果聚集相邻的U251细胞Notch-1及delta-like-1的平均荧光强度高于单个孤一的细胞。封闭后delta-like-1 mRNA的表达水平明显呈时间依赖性降低。结论在U251胶质瘤细胞中,Notch-1可能通过邻分泌机制促进相邻细胞Notch-1的表达,并影响其配体delta-like-1的表达。

Receptor role of membrane bound macrophage colony-stimulating factor

According to the definition of cytokine, the direction of signaling should be from cytokine to receptor. The counter receptor was presented. Membrane bound macrophage colony-stimulating factor (m-M-CSF) and its receptor (M-CSF-R) were shown in human leukemic cell line J6-1 as autojuxtacrine mechanism. Soluble M-CSF receptor (sM-CSF-R), which was isolated from J6-1 cells membrane, was added into J6-1 cell culture. It was observed inhibition of J6-1 cell proliferation, decreasing of mitosis index and ratio of multinuclear cells, enlargement of cell diameter and volume, down regulation of numerous surface antigens. Dramatic change of intracellular pH was shown between several min to 20 min after treatment of sM-CSF-R. It suggested that some information was transmitted via m-M-CSF from sM-CSF-R. This counter signaling was not influenced by saccharification of m-M-CSF.

支持细胞对体外培养精原干细胞的作用途径研究

目的：探讨支持细胞对体外培养精原干细胞的作用途径。方法：选用7 d龄雄性昆明种小鼠,两步酶消化法获得睾丸组织细胞悬液,差异时间贴壁法分离精原干细胞和支持细胞,免疫荧光法和油红O染色法分别对其进行生物学鉴定,流式细胞仪对精原干细胞进行纯度分析。按培养条件的不同将实验分为3组：精原干细胞与支持细胞共培养组（A组）、条件培养基组（B组）、常规培养基组（C组）。其中条件培养基按单纯支持细胞培养清液∶双倍浓缩的DMEM/F12∶胎牛血清=4.5∶4.5∶1的比列配置;常规培养基即含体积分数10%胎牛血清的DMEM/F12。台盼蓝法测定各组贴壁率,四甲基偶氮唑盐（MTT）法测定各组精原干细胞的吸光度并绘制增殖曲线。倒置显微镜下观察各组精原干细胞增殖特点及集落形成情况。比较各组精原干细胞24 h贴壁率、增殖曲线及存活时间的不同。结果：A组精原干细胞24 h贴壁率大于B组及C组（P〈0.05）,而B、C组间无差异（P〉0.05）;A组精原干细胞接种起即稳定增殖,于7~10 d形成稳定集落并维持约30 d的特性。B组和C组均表现为精原干细胞经短暂的增殖后呈现快速减少的趋势,培养1周后,精原干细胞数目明显减少。结论：支持细胞对体外精原干细胞的作用是依靠两者之间的直接联系和支持细胞的旁分泌2种途径;而仅依靠支持细胞的旁分泌作用不能促进精原干细胞的贴壁和增殖。

胰岛局部胰升血糖素分泌调节的研究进展

糖尿病的发生除了胰岛素分泌受损外,升糖激素胰升血糖素的分泌调节异常也参与其发生发展,且加重糖尿病的代谢结局。目前关于胰岛素分泌调节的研究已取得很大进展,但胰升血糖素分泌调节的研究较少。实际上,胰升血糖素除了受胰岛周围的旁分泌调节外,α细胞还具有自身的固有调节能力,且受邻近调节及钠葡萄糖同向转运体的调节。此外,胰升血糖素还受胰岛外因素的影响。因此,为更好地了解胰升血糖素复杂的分泌调节方式,进一步认识糖尿病的发生发展提供理论依据,本文拟对胰岛局部胰升血糖素分泌调节的研究进展做一综述。