绿色荧光转基因大鼠模型的建立

目的随着干细胞研究的推进,大鼠干细胞的研究日趋迫切。本研究旨在为活体荧光影像系统、干细胞归巢、细胞移植体内示踪研究,提供绿色荧光蛋白EGFP转基因大鼠模型。方法通过显微注射方式获得EGFP转基因大鼠,采用活体荧光影像系统、激光共聚焦显微镜,对EGFP转基因大鼠各个组织的荧光表达水平进行比较;采用流式细胞术检测转基因大鼠血液和骨髓细胞、骨髓干细胞的荧光标记率,筛选骨髓干细胞高效标记绿色荧光的转基因大鼠。结果建立了心脏、肝脏、肌肉、肺、胰腺、脑、膀胱、胃、肾脏、肠和脾脏组织中,系统性表达EGFP的SD-TgN（ACT-EGFP-1）ZLFILAS转基因大鼠;流式细胞术检测表明,该品系血液细胞绿色荧光标记率为94.4%,骨髓干细胞绿色荧光标记率为97.8%。结论建立了多组织系统性高表达绿色荧光,骨髓干细胞荧光标记率高达95%以上的转基因大鼠,为影像分析,造血干细胞的归巢等研究提供了大鼠模型。     

红色荧光和绿色荧光转基因小鼠模型的建立

目的建立红色荧光和绿色荧光转基因小鼠,为活体荧光影像系统建立重要的实验动物模型。方法把DsRed-Express和EGFP基因插入chicken-βactin强启动子下游构建转基因载体,建立红色荧光和绿色荧光转基因C57BL/6J小鼠。PCR鉴定红色荧光和绿色荧光转基因小鼠的基因表型,活体荧光影像系统分析红色荧光和绿色荧光转基因小鼠,荧光显微镜检测红色荧光和绿色荧光转基因小鼠全身组织器官的组织形态。结果分别建立了3个系的红色荧光和3个系的绿色荧光转基因小鼠。活体荧光影像系统分析转基因小鼠分别呈现红色荧光和绿色荧光。经荧光显微镜观察,DsRed-Express转基因小鼠的红色荧光蛋白在多个组织器官中表达,尤其在胰腺、肝脏、肾脏和脾脏等器官表达量较高。EGFP转基因小鼠绿色荧光蛋白在全身各个组织器官中表达,尤其在胰腺、心脏、小肠、外周血细胞和脑组织等器官组织中表达量较高。结论DsRed-Express和EGFP基因在转基因小鼠中系统性高表达,成功建立了红色荧光和绿色荧光转基因小鼠。DsRed-Express和EGFP转基因小鼠将成为活体荧光影像系统的重要实验动物模型。     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。     

3个不同品系小鼠ES细胞的绿色荧光蛋白标记

用电穿孔法将线性化的质粒pEGFP—N3分别导入来自129／ter、C57BL/6J和BALB／c3个品系的小鼠胚胎干细胞：MESPU—13、MESPU—35和MESPU—62中,经G418筛选、荧光显微镜镜检、阳性克隆扩增、流式细胞仪分选、再扩增以及核型分析等过程,分别得到核型大于85％的被EGFP稳定标记的细胞株5个(129／ter2个、C57BL/6J1个、BALB／c2个),分别命名为MESPU—13／G1和MESPU—13／G2、MESPU—35／G1、MESPU—62／G1和MESPU—62／G2。从不同品系中各选一个增殖生长快、形态典型的标记细胞株,进行碱性磷酸酶染色、oct4群基因产物的表达检测、类胚形成和体内分化鉴定,结果表明所得到的核型正常的、稳定标记的ES细胞系具有原ES细胞的典型特征。     

骨髓动员对骨髓造血干细胞在心肌梗死后心脏中定植及分化的影响

目的观察小鼠心肌梗死后骨髓造血干细胞在心脏内的分化及细胞因子的影响。方法C57/BL6小鼠60只分为骨髓动员组和对照组,先后行脾切除、骨髓移植（骨髓供体为增强绿色荧光蛋白转基因小鼠）、骨髓动员及建立心肌梗死模型。心肌梗死后3周将小鼠心脏取出并切片行组织学及激光共聚焦显微镜免疫荧光检查。结果骨髓动员可以增加EGFP阳性细胞在心脏中梗死区和边缘区的定植,但绝大多数EGFP阳性细胞都同时表达CD45。仅发现有极少数骨髓来源的心肌细胞、成纤维细胞及血管内皮细胞,且与骨髓动员无相关性。结论骨髓动员能够明显促进骨髓来源细胞定植入小鼠心脏的梗死区;极少数骨髓造血干细胞可以分化为心肌细胞,其数量远不足以修复梗死心肌及改善心功能;骨髓造血干细胞不参与梗死区疤痕形成的病理过程。     

EGFP-Luc-Hepa1-6细胞系的构建及其在小鼠肝癌模型中的应用

目的:构建带有增强型绿色荧光蛋白EGFP及荧光素酶luciferase的真核表达载体p CMVLuciferase-IRES2-EGFP,对肝癌细胞系Hepa1-6转染后进行稳定筛选,并将表达该载体的细胞系应用于小鼠原位肝癌模型构建,以对小鼠肝癌细胞进行稳定标记与活体示踪。方法:对p GL3-Basic质粒进行Xba I酶切、Klenow片段补平黏端、Xho I酶切获得luciferase小片段,与Xho I/Sma I酶切p IRES2-EGFP质粒获得的载体大片段连接,获得重组质粒p CMV-Luciferase-IRES2-EGFP。酶切鉴定、测序比对确认序列完全正确后,以重组载体转染Hepa1-6细胞进行稳定筛选,以荧光显微镜观察EGFP表达,报告基因实验与LuminaⅡ成像系统检测荧光素酶活性。确认该细胞系中的质粒得到表达后,以该细胞系进行C57BL/6小鼠肝脏原位接种构建肝癌模型,以LuminaⅡ成像系统连续活体监测肝癌的生长,取离体的肝癌组织制备石蜡切片(HE染色)和冰冻切片(免疫荧光染色)分别观察离体肿瘤组织病理学特征及其中肝癌细胞绿色荧光蛋白表达情况。结果:成功构建表达p CMV-Luciferase-IRES2-EGFP载体的Hepa1-6细胞系(EGFP-Luc-Hepa1-6),并以该细胞系成功构建C57BL/6小鼠原位肝癌模型,实现小鼠肝癌细胞的活体示踪与体外标记。结论:成功构建EGFP-Luc-Hepa1-6细胞系,且以此细胞系构建的小鼠原位肝癌模型可以同时实现小鼠肝癌生长的连续活体监测与离体组织检测。     

4个品系小鼠胚胎干细胞系的建立

探索高效的不同品系的小鼠胚胎干细胞的建系方法。B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠,孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG) 人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)促排,3.5天交配后(days post coitus,dpc)冲洗子宫取囊胚,或者2.5dpc冲洗输卵管,卵裂球体外培养获取囊胚。囊胚种植到小鼠成纤维细胞饲养层上干细胞培养液培养,4～5天内细胞团扩增后玻璃毛细管挑出,种植到新的饲养层上过夜再行胰蛋白酶消化,3～4天传代一次。对所建立的小鼠ES细胞系进行形态学、染色体核型、AKP染色、体内外分化能力,干细胞分子标记物荧光免疫染色等鉴定。获得10株小鼠胚胎干细胞,具有典型的胚胎干细胞生长特性,符合ES细胞的鉴定标准。结果表明成功的建立了来自B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠的10株ES细胞系。内细胞团挑出过夜增殖后消化的培养方法可能有助于提高ES细胞的建系率。     

C57BL／6J-HBV转基因小鼠血清生化指标与性别年龄的关系

目的观察C57BL/6J-HBV乙型肝炎病毒转基因小鼠血清总胆红素（T-BIL）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、总蛋白（TP）和白蛋白（ALB）与性别和年龄的关系,以及与遗传背景相同的C57BL／6J小鼠的差异。方法选取8周龄和24周龄的C57BL／6J-HBV转基因小鼠及C57BL／6J小鼠的血清测定T-BIL、ALT、AST、TP和ALB值。结果C57BL／6J-HBV转基因小鼠与同周龄同性别的C57BL／6J小鼠相比,T-BIL、ALT、AST、TP和ALB均存在显著差异（P〈0．05）;24周龄的C57BL／6J-HBV转基因小鼠ALT和AST与其8周龄鼠相比均存在显著差异（P〈0．05）。结论C57BL／6J-HBV转基因小鼠T-BIL、ALT、AST、TP和ALB值显著高于C57BL／6J小鼠;且C57BL／6J-HBV转基因小鼠ALT和AST值与年龄有关,与性别无关。     

绿色荧光裸鼠的建立和验证

目的建立系统性表达绿色荧光蛋白的裸鼠,接种人源肺癌细胞验证该模型是否具有免疫缺陷性,并观察双色荧光的成像效果。方法利用系统性表达绿色荧光蛋白的C57BL/6J小鼠与BALB/C裸小鼠多代杂交和互交,建立稳定表达绿色荧光蛋白的裸鼠。大体解剖观察胸腺生长情况,整体和器官荧光成像验证绿色荧光蛋白的表达情况。以2×106/只的剂量对其皮下腋下接种表达红色荧光蛋白的人类A549肺癌细胞（RFP-A549）,通过观测肿瘤生长来验证模型的免疫缺陷性。同时,利用红色荧光标记的肿瘤和绿色宿主鼠,对双色的整体成像效果进行观测。结果构建出系统性表达绿色荧光蛋白的裸鼠,大体解剖可见胸腺缺失。在激发光的激发下,绿色荧光裸鼠全身发出清晰的绿色荧光,脑、心脏、肺脏、肝脏、肾脏,肠胃及胰腺等主要器官可见明显绿色荧光。接种RFP-A549细胞后,成瘤率达到100%,整体动物荧光成像表现出清晰的双色。结论本研究构建出的绿色荧光裸鼠,动物整体可以清晰地表达绿色荧光并具有免疫缺陷性     

不同小鼠品系囊胚受体对C57BL/6胚胎干细胞种系嵌合效率的影响

目的考察不同品系的小鼠囊胚对C57BL/6胚胎干细胞种系嵌合效率的影响,进而提高通过在C57BL/6胚胎干细胞中同源重组制作基因打靶小鼠的效率。方法选取近交系B6(Cg)-Tyrc-2J和BALB/c品系及封闭群ICR作为囊胚供体鼠,通过在TLX3、Ai3K和SL三个不同基因修饰ES细胞的种系嵌合实验中,平行比较三者的囊胚利用率、嵌合鼠获得效率以及种系遗传效率等三个方面,并进一步针对囊胚来源这一因素对效率的影响进行统计学分析。结果三种ES细胞均未能通过B6(Cg)-Tyrc-2J品系囊胚的注射获得种系遗传。而BALB/c品系与ICR品系相比,囊胚利用率明显低于ICR品系(P0.05);嵌合鼠获得效率方面,BALB/c与ICR相当(P=0.115);而种系遗传效率方面,BALB/c品系显著高于其他品系(P0.01)。结论 BALB/c品系在C57BL/6胚胎干细胞种系嵌合效率方面确实具有优势,然而,由于BALB/c囊胚较难获得,并且存在发育延迟和透明带脆性高等问题,而ICR品系在囊胚获得和利用方面的效率明显高于BABL/c,并且也可以支持C57BL/6胚胎干细胞的种系遗传,因此也是C57BL/6胚胎干细胞囊胚注射中较好的选择。     

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.     

人尿激酶型纤溶酶原激活因子截短型突变体与绿色荧光蛋白在真核细胞HEK293F中的融合表达

目的:构建人尿激酶型纤溶酶原激活因子(uPA)截短型突变体与绿色荧光蛋白(EGFP)分泌型融合表达载体并在真核细胞中表达。方法:采用PCR法,分别以质粒pIRES2-EGFP和重组质粒pcDNA3.1(+)/uPA为模板,扩增出带BamHⅠ和XbaⅠ酶切位点的EGFP及带NheⅠ和HindⅢ酶切位点的uPA截短体基因片段,先后将EGFP和截短型uPA基因片段克隆到真核表达载体pcDNA3.1(+)上,转入HEK293F细胞,用G418对转染细胞进行加压筛选,通过共聚焦显微镜观察和ELISA方法鉴定表达产物。结果:DNA测序结果显示,uPA不同截短型突变体基因片段与EGFP基因融合的真核表达载体构建成功,共聚焦显微镜观察发现HEK293F细胞中有绿色荧光且定位于细胞质中,ELISA检测到HEK293F细胞培养上清中分泌型融合蛋白的表达。结论:构建了uPA截短型突变体与EGFP分泌型融合表达载体并在真核细胞中表达,为后期研究uPA的相互作用蛋白及其生理功能奠定了基础。     

The positive regulatory effect of TGF-beta2 on primitive murine hemopoietic stem and progenitor cells is dependent on age, genetic background, and serum factors

TGF-beta is considered a negative regulator of hemopoietic stem and progenitor cells. We have previously shown that one TGF-beta isoform, TGF-beta2, is, in fact, a positive regulator of murine hemopoietic stem cell function in vivo. In vitro, TGF-beta2, but not TGF-beta1 and TGF-beta3, had a biphasic dose response on the proliferation of purified lin-Sca1(++)kit(+) (LSK) cells, with a stimulatory effect at low concentrations, which was subject to mouse strain-dependent variation. In this study we report that the stimulatory effect of TGF-beta2 on the proliferation of LSK cells increases with age and after replicative stress in C57BL/6, but not in DBA/2, mice. The age-related changes in the TGF-beta2 effect correlated with life span in BXD recombinant strains. The stimulatory effect of TGF-beta2 on the proliferation of LSK cells requires one or more nonprotein, low m.w. factors present in fetal calf and mouse sera. The activity of this factor(s) in mouse serum increases with age. Taken together, our data suggest a role for TGF-beta2 and as yet unknown serum factors in the aging of the hemopoietic stem cell compartment and possibly in organismal aging.     

乙型肝炎病毒蛋白和绿色荧光蛋白的共表达研究

目的：构建增强型绿色荧光蛋白（EGFP）标记的乙型肝炎病毒（HBV）真核表达载体,并研究其在真核细胞和小鼠体内的共表达。方法：以质粒pBR322-HBVadr2．0和pCX-EGFP为基础,构建含有双拷贝HBV全基因组DNA和EGFP基因的真核表达载体pCX-EGFP-HBVadr2．0,分别转染真核细胞和小鼠肝组织,建立体外、体内表达系统,研究GFP和HBV基因的表达。结果：构建了真核表达载体pCX-EGFP-HBVadr2．0,EGFP和HBV病毒蛋白在体内和体外均可表达。结论：构建的pCX-EGFP-HBVadr2．0真核表达载体可以GFP作为HBV存在与否的报告基因,提高了培育检测转基因小鼠的效率,为转基因小鼠的制备及后续研究奠定了基础。     

FISH analysis of 142 EGFP transgene integration sites into the mouse genome

Production of transgenic animals is an important technique for studying various biological processes. However, whether the integration of a particular transgene occurs randomly in the mouse genome has not been determined. Analysis by fluorescence in situ hybridization of the integration sites of the 142 EGFP (a mutant of green fluorescent protein) transgenic lines that we produced showed that the transgenes had become incorporated into every mouse chromosome. A single integration site was observed in 82.4% of the lines. The concomitant integrations of transgene into two different loci were observed in 15 cases (10.6%). In 3 cases, the transgenic founder mice showed chimerism in integration sites (2.1%). Chromosomal translocation was observed in 7 cases (4.9%). Moreover, when we statistically analyzed the transgene integration sites of these mouse lines, they were shown to distribute unevenly throughout the genome. This is the first report to analyze the transgene integration sites by producing more than 100 transgenic mouse lines.     

小鼠骨片间充质干细胞分离培养及鉴定

目的建立小鼠骨片间充质干细胞（MSC）分离培养及扩增的方法。方法取小鼠胫骨和股骨,洗去骨髓后,用胶原酶I消化疏松骨密质,利用MSC具有迁徙和贴壁生长的能力进行分离。并对获取的细胞进行流式鉴定和诱导分化。结果培养2d小鼠骨片边缘爬出成纤维样细胞,呈克隆和鱼群样生长,并可以进行持续传代培养。流式鉴定结果显示这群细胞表达MSC标志Scall（92．7％）,CD29（98．4％）,CD90（91．6％）,不表达造血细胞标志CD34（1．57％）,CD45（3．99％）,CD11b（0．63％）,并可成功诱导分化成骨细胞和脂肪细胞。结论成功建立从小鼠骨片中获得MSC的方法,为实验研究提供可靠的细晌实源．     

In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy

Animal imaging requires the use of reliable long-term fluorescence methods and technology. The application of confocal imaging to in vivo monitoring of transgene expression within internal organs and tissues has been limited by the accessibility to these sites. We aimed to test the feasibility of fibred confocal fluorescence microscopy (FCFM) to image in situ green fluorescent protein (GFP) in cells of living animals. We used transgenic rabbits expressing the enhanced GFP (eGFP) gene. Detailed tissue architecture and cell morphology were visualised and identified in situ by FCFM. Imaging of vasculature by using FCFM revealed a single blood vessel or vasculature network. We also used non-transgenic female rabbits mated with transgenic males to visualise eGFP expression in extra-foetal membranes and the placenta. Expression of the eGFP gene was confirmed by FCFM. This new imaging technology offers specific characteristics: a way to gain access to organs and tissues in vivo, sensitive detection of fluorescent signals, and cellular observations with rapid acquisition at near real time. It allows an accurate visualisation of tissue anatomical structure and cell morphology. FCFM is a promising technology to study biological processes in the natural physiological environment of living animals.     

Characterizations of the murine mesenchymal cell line expressing GFP

We established and characterized a murine mesenchymal stem cell line from the bone marrow of a transgenic C57BL mouse that ubiquitously expressed green fluorescent protein (GFP). Immunostaining revealed the presence of several markers common for mesenchymal stem cells (MSCs). The cells expressed specific fibroblast proteins, such as smooth muscle actin, which is localized in stress fibrils, and vimentin, a major protein of intermediate filaments in connective tissue cells. These proteins are responsible for the ability to differentiate into adipocytes or osteoblasts under appropriate conditions. The MSC karyotype was unstable. At the 6th passage cells, were aneuploid and genetically heterogeneous. The number of chromosomes ranged from near 2n to 8n. 80% of cells had chromosome numbers between 50 and 85 without a well-defined modal class. Differential G-staining of metaphase spreads showed variability in the copy numbers of individual chromosomes and presence of random chromosome rearrangements, such as ectopic associations of nonhomologous chromosomes. All cells analyzed contained a single dicentric marker chromosome. Some cells also had mini-chromosomes regarded as indicators of gene amplification. We suppose that the karyotypic instability of MSCs that express GFP is provoked by the insertion of foreign GFP transgenes into the murine genome. These cells could be useful for the study of genomic alterations during the spontaneous oncogenic transformation of stem cells.