Gene Transfer into Hematopoietic Cells of Mouse and Its in vivo Expression after Transplantation

Genetransferintohematopoieticcellshasfoundmanyapplicationinexperimentalresearchesaswellasclinicalpractice['1.Atpresent,thestrategyofgenetransferusedmostcommonlyistheretrovirusvectorsystem[2].However,theuseofretrovirusvectorssuffersfromseveraldrawbacks:(1)thegenerationandmanufacturingofretrovirusvectorsarecomplexandnotinexpensive;(2)generalsafetyconcernsareassociatedwiththeuseofanyviral--basevectorsystem,includingreplicationdefectivemurineretrovirusvectorsj(3)retrovirusvectorscurrentlyusedareab…     

Gene transfer into hematopoietic cells of mouse and itsin vivo expression after transplantation

Summary We have shown previously that high-efficient gene transfer can be attained in primary hematopoietic cells using liposome-mediated gene transfer strategy. In order to examine the stability of gene expression mediated by this gene transduction protocol, we observed the expression of marker genein vivo by using bone marrow transplantation (BMT) to engraft lethally irradiated mouse with the genetically modified hematopoietic cells. The results showed that the mouse transplanted with appropriated number of transduced cells remained alive and healthy. The PCR analysis and G418 selection of the spleen colonies and bone marrow cells isolated from lethally irradiated animals 15 days and 30 days after injection of genetically modified bone marrow cells showed that the progeny cells of the transduced hematopoietic stem cells still contained and expressed the transduced genes, suggesting that the hematopoietic system is at least partially re-constructed by the stem cells with marker gene and that the stable expression of foreign genesin vivo can be attained by using this easy and harmless transduction protocol. These findings provide experimental basis for clinician to further investigate the biology of marrow reconstruction and the mechanism of leukemia relapse after BMT. This project was supported by the grant of Scientific Research Foundation of Public Health Ministry (320. 2430-330).     

小鼠骨髓细胞肾上腺素β2受体的表达及其意义

目的：探讨骨髓细胞肾上腺素β₂受体(β₂-AR)的表达及其意义,为神经因素调节骨髓造血理论提供依据。方法：用Ficoll淋巴细胞分离液从正常小鼠骨髓分离的单个核细胞直接涂片、粒-单集落造血祖细胞培养后涂片、骨髓基质细胞培养爬片,同时行骨髓石蜡切片及全骨髓细胞涂片,用免疫组织化学SP法检测上述标本中骨髓细胞β₂-AR的表达。另取小鼠采用淋巴细胞注射法制备免疫介导的再生障碍性贫血（简称再障）模型,β₂-AR拮抗剂组静脉注射ICI 118551,对照组静脉注射生理盐水,至第8天采用Western blotting 检测3组小鼠骨髓单个核细胞β₂-AR的表达。结果：正常小鼠骨髓细胞β₂-AR免疫组织化学反应可见胞质及胞膜呈深浅不等的棕黄色,其中单个核细胞48%强阳性,造血祖细胞55%中等阳性、45%弱阳性,骨髓基质细胞68%强阳性,其他辅助细胞呈不同程度的阳性反应。再障模型组、β₂-AR 拮抗剂组及对照组小鼠骨髓单个核细胞β₂-AR相对表达量分别为1.03±0.31、1.72±0.29及1.41±0.35。与对照组比较,再障模型组β₂-AR表达量明显降低（P<0.05）,β₂-AR拮抗剂组β₂-AR表达明显增加（P<0.05）。结论：正常小鼠骨髓细胞有β₂-AR表达,其表达量与骨髓造血状态有关,提示β₂-AR可能通过神经-内分泌-免疫网络途径参与造血调节。     

Chondrogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells Induced by Cartilage-derived Morphogenetic Protein-2 In Vitro

To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 （0, 10, 20, 50 and 100 ng/mL）. After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen Ⅱ mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen Ⅱ mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.     

重组β-半乳糖苷酶腺病毒载体的构建及其在血管平滑肌细胞中的表达

目的 :构建表达 β -半乳糖苷酶 (β -gal)的重组腺病毒 ,以期为再狭窄的基因治疗研究提供一个对照载体和为腺病毒介导的基因转移的安全性、可行性、转染效率的研究提供一有用的工具。方法 :采用基因重组方法构建穿梭质粒pAd - β -gal,通过同源重组法制备出重组腺病毒Ad - β -gal,转染的 2 93细胞用X -gal染色鉴定Ad - β-gal的正确与否。通过测定 2 6 0nm的紫外光吸收值估计病毒高度。原代大鼠血管平滑肌细胞采用组织块培养法。Ad - β -gal以 10 0moi感染VSMCs并行X -gal染色 ,以观察 β -gal在VSMCs中的表达情况。 结果 :成功构建了pAd - β -gal穿梭质粒和Ad - β -gal重组腺病毒 ,转染的 2 93细胞和VSMCs经X -gal染为蓝色 ,病毒浓度为 9× 10 11pfu/ml,以 10 0moiAd - β-gal转染VSMCs,其转染效率接近 10 0 %。结论 :表达 β -gal的重组腺病毒构建成功 ,并在VSMCs中得到有效表达 ,本研究为将来再狭窄基因治疗研究提供了一对照载体 ,也为腺病毒介导的基因转移的安全性、可行性、有效性的研究提供了一有用的工具。     

bcl-2基因转染小鼠造血干细胞重建造血的生物学特性

目的 观察ｂｃｌ－２基因转染小鼠造血干细胞重建造血的小鼠对放化疗的抵抗作用。方法 （１）含ｂｃｌ－２ｃＤＮＡ的逆转录病毒载体质粒,经脂质体介导转染ＰＡ３１７包装细胞,通过Ｇ４１８筛选和克隆,获得高病毒滴度的细胞株（Ｄ３）;（２）用Ｄ３细胞株的病毒上清转染小鼠胚胎肝造血干细胞;（３）经致死剂量照射小全注ｂｃｌ－２转 造血干细胞重建造血;（５）采用脾集落形成和再增殖能力观察ｂｃｌ－２转因干细胞重建造血     

Chemoprotection of transfer of multidrug resistance gene into human hematopoietic progenitor cell

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转基因溶瘤性单纯疱疹病毒对人类乳腺癌细胞及其干细胞的溶瘤作用

 【目的】 通过体外实验观察溶瘤性单纯疱疹病毒(HSV)载体G47Δ对人类原代乳腺癌细胞及乳腺癌干细胞毒效应,探讨其对人乳腺癌的治疗作用。【方法】 选取6例浸润性导管癌组织,体外培养人原代乳腺癌细胞及人原代乳腺癌干细胞,将G47Δ按不同感染复数(MOI),即MOI=0.01和MOI=0.1加入各实验组中,观察每天细胞的生长情况。G47Δ含有LacZ基因,其表达产物可被X-gal染色检测。 【结果】 G47Δ病毒在乳腺癌细胞中可复制和传播通过X-gal染色法得以证实。 G47Δ对人原代乳腺癌细胞及乳腺癌干细胞都具有杀伤作用,对乳腺癌细胞更加敏感。在MOI=0.01组及0.1组中,感染病毒后第4 d,人原代乳腺癌细胞被杀灭的比例分别是91%及96%,乳腺癌干细胞被杀灭的比例分别是43%及78%;感染病毒后第6天,两组中人原代乳腺癌细胞接近全部被杀灭,乳腺癌干细胞被杀灭的比例分别是92%、98%。 【结论】 G47Δ可有效杀灭人乳腺癌细胞及其干细胞;乳腺癌细胞比乳腺癌干细胞对G47Δ更具有敏感性。     

无辅助病毒HSV-1载体介导LacZ基因在培养皮层神经元中的表达

目的:探讨无辅助单纯疱疹病毒(herpes simplex virus type-1,HSV-1)载体的包装及其介导LacZ基因在体外培养神经元表达的作用, 解决该载体系统在包装及体外培养神经元应用中的有关问题.方法:将一组含HSV-1基因组的粘粒以限制性内切酶Pac Ⅰ酶切、纯化后,与含LacZ和酪氨酸羟化酶(tyrosine hydroxylase,TH)及神经微丝(neurofilament,NF)嵌合启动子的HSV-1质粒DNA在脂质体作用下,共转染2-2细胞;在34 ℃条件下培养72 h,收获病毒颗粒.将含病毒颗粒的上清液,加入BHK细胞培养液中,继续培养24 h后,加X-gal染液作用4 h,观察表达LacZ基因的蓝染细胞.取培养第3天的胚胎大鼠大脑皮层神经元,加入含病毒颗粒的培养液,过夜后更换新鲜培养液,分组继续培养1,7和14 d,进行X-gal染色,光镜下观察.结果:包装形成的病毒颗粒感染BHK细胞,24 h后可见表达LacZ基因的蓝染细胞;感染培养第3天的皮层神经元,继续培养1,7和14 d后均可见蓝染神经元.结论:无辅助病毒包装系统能够使含TH-NF嵌合启动子的HSV-1载体形成有效的病毒颗粒,并且在体外培养神经元中持续稳定地表达外源基因,为在神经系统进行基因转移和基因功能研究提供了有效的工具.     

转LacZ基因骨髓间充质干细胞体内造血分化的初步研究

目的　初步探讨小鼠骨髓间充质干细胞在体内造血细胞分化的潜能。方法　利用转LacZ基因小鼠MSCs输注X射线亚致死量照射的BALB/C小鼠,2个月后取骨髓细胞作体外甲基纤维素培养和X -Gal染色,取肝脏X -Gal染色。结果　MSCs输注组和对照组骨髓细胞体外集落培养均能形成鹅卵石样造血细胞集落,经X -gal组化染色后,输注组部分造血集落产生蓝绿色,对照组没有颜色变化。肝脏X -Gal染色输注组部分产生蓝色,对照组没有颜色变化。结论　小鼠MSCs具有向造血细胞分化的潜能。     

敲除PKD1基因的小鼠胚胎干细胞的建立

目的：通过PKD1基因打靶载体的胚胎干细胞转染，药物筛选以及分子鉴定，获得发生同源重组的胚胎干细胞克隆，为产生PKD1基因缺陷小鼠品系准备条件。方法：体外培养小鼠胚胎干细胞，用电穿孔的方法进行PKD1基因打靶载体的转移，并用G418进行筛选培养，得到阳性克隆后，进一步扩大培养，抽提胚胎干细胞的基因组DNA，采用DNA印迹法进行分子鉴定。结果：经G418筛培养得到192个阳性在隆，分子鉴定获得2个发生同源重组的胚胎干细胞克隆。结论：鉴定得到的2个同源重组胚胎干细胞克隆，为进一步建立PKD1基因缺陷小鼠奠定了基础。     

小鼠骨髓中性粒细胞分离纯化及活性检测研究

目的 探究小鼠骨髓中性粒细胞分离纯化,并进行鉴定以及活性的检测。方法 使用密度梯度离心法分离纯化小鼠骨髓中性粒细胞,得到的中性粒细胞采用免疫荧光法和高内涵分析仪检测中性粒细胞表面标志物Ly6G的表达。同时对得到的细胞加入佛波酯（phorbol 12-myristate 13-acetate,PMA）刺激后,采用免疫荧光的方法检测中性粒细胞释放髓过氧化物酶（myeloperoxidase,MPO）的表达情况。结果 用Histopaque^©-1077、Histopaque^©-1119分离纯化小鼠骨髓细胞后,细胞表达Ly6G,将分离纯化的细胞加入100 nmol/L PMA刺激2 h后检测MPO的表达增强,实验组是对照组的（3.26±0.22）倍。结论 该方法是一种简单、易操作的体外分离纯化、鉴定小鼠骨髓中性粒细胞的方法。     

新生大鼠雪旺细胞的体外培养和纯化

目的:探索简单有效获取大量高纯度雪旺细胞的新方法。方法:分离出生3d的新生SD大鼠坐骨神经,使用胰蛋白酶及I型胶原酶混合消化,结合差速贴壁法和G418纯化。四氮唑盐(MTT)比色法测定细胞的生长增殖情况,免疫细胞化学方法计算雪旺细胞的纯度。结果:胰酶和胶原酶混合消化后的活细胞率为94.6±3.4%,纯化后的雪旺细胞纯度为95.6±2.5%。结论:胰酶和胶原酶混合消化结合差速贴壁和G418纯化是获取大量高纯度雪旺细胞的简单有效方法。     

Fth1基因标记对骨髓间充质干细胞生物学特性及体外MRI表现的影响

摘要 目的: 观察铁蛋白报告基因Fth1标记对骨髓间充质干细胞(MSCs)生物学特性的影响及体外MRI表现。方法：原代分离培养大鼠MSCs,构建携带铁蛋白重链基因Fth1的慢病毒载体,取第4代MSCs进行转染,并在培养基内加入柠檬酸铁进行培养。普鲁士蓝染色检测转染MSCs的摄铁能力,台盼蓝染色活细胞计数检测转染细胞的活力,CCK-8测定转染MSCs的增殖活性。应用MRI的FSE T2WI和SWAN序列观察转染MSCs和普通MSCs MRI信号的差异。结果：Fth1基因可成功转染MSCs,转染MSCs的普鲁士蓝染色标记效率为87%;未加含铁培养液的转染MSCs,活细胞比率为92.17±1.91,OD值为1.094±0.0682,与普通MSCs对照组比较差异均无统计学意义（P>0.05）,加入含铁培养液培养3天后的转染MSCs活细胞比率为77.47±4.10,OD值为0.4929 ±0.0239,与未加含铁培养液的标记MSCs及对照组比较差异均有统计学意义（P<0.05）;MRI扫描FSE T2WI和SWAN序列显示转染MSCs信号强度为847.1±10.54,与未加含铁培养液的转染MSCs及对照组比较差异均有统计学意义(P<0.05),而未加含铁培养液的转染MSCs与对照组比较差异无统计学意义（P>0.05）。结论：Fth1报告基因可成功转染MSCs、并高效表达与摄取铁,不影响细胞活力及增殖活性,但在铁浓度1mol/L含铁培养液中细胞活性受到一定影响;经含铁培养液培养5天后,MRI可体外检测标记MSCs,其FSE T2WI和SWAN序列呈低信号改变。     

Expression of X-linked Inhibitor of Apoptosis Protein and Its Effect on Chemotherapeutic Sensitivity of Bladder Carcinoma

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemothera- peutic sensitivity of T24 cells.     

反转录病毒载体介导的人β—珠蛋白基因在小鼠体内整合和表达的？…

OBJECTIVE: To examine the in vivo properties of retroviral recombinants carrying partially deleted human beta-globin gene (delta beta) and truncated erythroid enhancer (292 bp and 341 bp of 5'HS2) at the mRNA levels following short- and long-term reconstitution in mice with infected marrow cells. METHODS: First ecotropic virus producer cell lines with higher virus titers were isolated using "ping-pong" procedures. Then the human beta-globin gene was transferred into murine hematopoietic progenitor cells and the integration and expression of transferred gene were analyzed by southern blot and RNase protection assay or RT-PCR. RESULTS: The virus titers of both recombinants increased obviously after "ping-pong" procedures. The transferred human beta-globin gene was detected in murine CFU-S12 and the expression level was about 0.5%-5% of endogenous mouse alpha-globin gene. In 3 of 14 mice surviving long-term transplanted with bone marrow cells transduced with high-titer virus, bone marrow, spleen and thymus from two mice and bone marrow and spleen from another mouse contained the intact proviral genome. Long-term expression of the transferred gene was seen in one mouse at level of 7% of endogenous murine alpha-globin gene. CONCLUSIONS: The transferred human beta-globin gene can stably integrate into murine hematopoietic stem cells mediated by retroviral vectors and express in an erythroid-specific manner.     

B7基因转染骨髓干/祖细胞及表达的实验研究

目的探索非实体瘤白血病转基因治疗的可能性,以便临床应用.方法利用脂质体为载体,将B7基因转导入骨髓干/祖细胞内,通过G418筛选,收集转导的阳性细胞;以CD80单克隆抗体免疫荧光标记检测,观察B7基因在人骨髓干/祖细胞内基因转移及其表达的稳定性.结果通过脂质体介导,B7基因在人的骨髓细胞可以有效的稳定表达;用G418筛选可使转导的阳性细胞富集.结论利用脂质体做载体介导骨髓造血干/祖细胞转移B7基因是可行的.     

Liposome-mediated functional expression of multiple drug resistance gene in human bone marrow CD34<Superscript>+</Superscript> cells

Summary The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34⁺ cells was observed. Human normal bone marrow CD34⁺ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34⁺ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34⁺ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34⁺ cells was obviously higher than that in non-transfected CD34⁺ cells. The amount of P-gp in non-transfected CD34⁺ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34⁺ cells. CAO Wenjing, female, born in 1968, Doctor in Charge     

携带野生型PTEN基因的重组腺病毒治疗子宫内膜癌的体外研究

目的:观察携带野生型PTEN基因的重组腺病毒(Ad-PTEN)体外转染子宫内膜癌细胞后的表达,并探讨其对肿瘤细胞产生抑制增殖、诱导凋亡的作用及机制.方法:细菌内同源重组方法构建Ad-PTEN,体外转染PTEN基因突变的子宫内膜腺癌RL95-2细胞中,X-gal染色检测转染效率.应用RT-PCR、Western印迹、细胞免疫组化检测Ad-PTEN在RL95-2细胞中的转录及表达; 应用细胞计数、MTT实验, 观察Ad-PTEN对RL95-2细胞生长的影响;应用光镜、透射电镜观察外源性PTEN基因表达对RL95-2细胞形态学及超微结构的影响;应用流式细胞分析仪(FCM)检测Ad-PTEN对RL95-2细胞周期分布的影响、凋亡诱导作用及半胱氨酸天冬氨酸特异蛋白酶casapase-3的激活.结果:外源性野生型PTEN基因经腺病毒介导成功转入RL95-2细胞,3种方法均检测出有PTEN mRNA及PTEN蛋白的表达,当感染复数(MOI)为50时,体外转染效率达到100%.Ad-PTEN显著抑制RL95-2细胞生长并诱导其凋亡.此外, Ad-PTEN还能诱导RL95-2细胞周期G0/G1期阻滞以及caspase-3的激活.结论:重组腺病毒Ad-PTEN是高效的基因转移系统,能将PTEN目的基因转移到丧失该基因功能的子宫内膜癌RL95-2细胞中,对RL95-2细胞产生强有力的生长抑制及凋亡诱导作用,其机制可能包括细胞周期G0/G1阻滞及caspase-3 蛋白酶的激活.