应用转基因小鼠模型研究细小病毒非结构基因抗肿瘤作用

目的探讨小鼠细小病毒非结构基因转基因小鼠抗肿瘤的作用。方法给予肺组织表达非结构（ＮＳ）基因的首建者转基因小鼠Ａ６子代雄性鼠腹腔注射氨基甲酸乙酯水溶液,以诱发肺瘤结节。给予肩胛皮下不表达ＮＳ基因的Ａ６子代雌性鼠局部注射致癌剂３┐甲基胆蒽苯溶液,以诱发肩胛皮下肿瘤,另一部分子代雌性鼠腹腔接种Ｓ１８０肉瘤细胞２×１０６个／只,使之荷瘤。结果诱发携带ＮＳ基因组小鼠肺瘤结节平均数显著少于未携带ＮＳ基因组（Ｐ＜０．０１）。携带与未携带ＮＳ基因小鼠组的肩胛皮下肿瘤形成率、平均瘤重无显著差异（Ｐ＞０．０５）。腹腔接种Ｓ１８０肉瘤细胞小鼠,携带与未携带ＮＳ基因组的生命延长率无显著差异（Ｐ＞０．０５）。结论表达ＮＳ基因的组织具有抑制化学致癌剂致瘤形成的作用,而不表达ＮＳ基因的组织则无此作用,转基因小鼠对外源性肿瘤细胞的生长无抑制作用。ＮＳ基因可能主要在表达的细胞内发挥作用,抑制细胞的恶性转化     

乙肝病毒（Adr）全基因组在转基因小鼠体内（HBV）DNA的存在状态

本文用ＰＣＲ，ＳｏｕｔｈｅｒｎＢｌｏｔ方法检测了３４只Ｇｏ代２６只转基因小鼠携带ＨＢＶＤＮＡ（阳性率为７６％），５１５只Ｇ１代转基因小鼠中２４８只组织中携带ＨＢＶＤＮＡ（阳性率为４８％），２５只Ｇ２代转基因小鼠中１８只组织中携带ＨＢＶＤＮＡ（阳性率为７２％）．对１０８只尼组织阳性小鼠血清检测，１０２只小鼠血清呈ＨＨＶＤＮＡ阳性。以上结果证明，ＨＢＶＤＮＡ基因已整合小鼠的染色体中。这些转基因小鼠对研究ＨＢＶ病毒的复制与表达，慢性肝炎以及肝癌发生的机理，治疗乙肝药物的筛选等具有十分重要的价值。     

HSV-tk/GCV协同放射治疗黑色素瘤的实验研究 *

目的：了解ＨＳＶ－ｔｋ／ＧＣＶ系统协同放射治疗对小鼠黑色素瘤的联合杀伤作用。方法：通过逆转录病毒介导,将潮霉素磷酸转移酶和ＨＳＶ－ｔｋ的融合基因（ｈｙｔｋ）导入黑色素瘤细胞中并获得表达,通过体外和Ｃ５７ＢＬ／６小鼠动物实验,检测 ＧＣＶ对转基因细胞的体内外杀伤作用及联合应用放射治疗对黑色素瘤的治疗作用。结果：ＰＣＲ、ＲＮＡ Ｄｏｔ ｂｌｏｔｔｉｎｇ、免疫组化检测证实ＨｙＴＫ在转基因细胞的表达,体内外实验示ＧＣＶ对Ｂ１６／ＨｙＴＫ细胞有明显的杀伤作用,而对野生型Ｂ１６细胞无明显杀伤作用（Ｐ＜０．０５）;联合应用低剂量ＧＣＶ可使转入ｈｙｔｋ基因的黑色素瘤细胞对放疗的敏感性明显增高（ＳＥＲ＝１．６２）,体内实验也证实协同疗法可延缓荷瘤小鼠的肿瘤生长。结论：ＨＳＶ－ｔｋ／ＧＣＶ系统协同放射治疗有望成为黑色素瘤基因治疗的一种有效方法。     

非小细胞肺癌p16基因突变的初步研究

为了解非小细胞肺癌（ＮＳＣＬＣ）ｐ１６基因突变的情况,本研究应用ＰＣＲ－ＳＳＣＰ银染技术检测了２４例ＮＳＣＬＣ肿瘤组织中ｐ１６基因外显子２的突变情况,现将结果报告如下。１　材料与方法２４例ＮＳＣＬＣ肿瘤组织及同一病例非肿瘤肺组织均为住院病例手术切除标本,并经病理证实。按常规方法提取ＤＮＡ。ＰＣＲ反应在ＰＥ９６００热循环仪中进行,扩增ｐ１６基因第２外显子,引物序列：　　ＰＳ１：５′－ＡＣＡＡＧＣＴＴＣＣＣＴＴＣＣＧＴＣＡＴＧＣ－３′ＰＳ２：５′－ＴＣＴＧＡＧＣＴＴＴＧＧＡＡＧＣＴＣＴＣＡＧ－３′循…     

乳腺癌组织BRCA1,BRCA1基因区域杂合性缺失的研究

目的 研究乳腺癌组织ＢＲＣＡ１、ＢＲＣＡ２基因区域的杂合性缺失（ＬＯＨ）情况。方法 采用聚合酶链式反应（ＰＣＲ）和聚丙烯酰胺凝胶电泳,对３０例散发性乳腺癌和２例姐妹乳腺癌进行ＬＯＨ的检测,同时还采用Ｇ显带法对姐妹乳腺癌患者的外周血淋巴细胞染色体畸变情况进行分析。结果 在散发性乳腺癌中,ＢＲＣＡ１、ＢＲＣＡ２基因区域的杂合性缺失率分别为６．１２％和５．７７％;姐妹二人乳腺癌组织在ＢＲＣＡ２基因区域的     

2（3）—叔丁基—4—羟基茄香醚对HBV转基因小鼠肝肿瘤的防护作用研究

目的 观察抗氧化剂２（３）－叔丁基－４－羟基茄香醚（ＢＨＡ）对ＨＢＶ转基因小鼠肝肿瘤的防护作用。方法２６只ＨＢＶ转基因和２４只非转基因小鼠,用生化法测定肝脏混还原酶（ＱＲ）和谷胱甘肽Ｓ－转移酶（ＧＳＴｓ）内二醛（ＭＤＡ）含量和免疫组化法研究肝肿瘤ＰＣＮＡ表达。结果 转基因小鼠ＨＢＡ组肝癌发生率０,显著低于普饲组４２％。ＢＨＡ诱导肝脏ＱＲ和ＳＴ活性升高３－７倍,降低ＭＤＡ。ＢＨＡ组肝腺瘤ＰＣＮＡ标准     

复制型HBV（adr）转基因小鼠G_2、G_3代血清中HBV的表达──电镜检测

复制型ＨＢＶ（ａｄｒ）转基因小鼠Ｇ_２、Ｇ_３代血清中ＨＢＶ的表达──电镜检测前已报道：上海市肿瘤研究所与扬州大学江苏农学院已成功地建立了复制型ＨＢＶ（ａｄｒ）转基因小鼠模型（肿瘤，１９９４，１４：１８６），经ＰＣＲ，Ｓｏｕｔｈｅｒｎｂｌｏｔ方法检测证...     

2(3)-叔丁基-4-羟基茴香醚对HBV转基因小鼠肝肿瘤的防护作用研究

观察抗氧化剂２（３）┐叔丁基┐４┐羟基茴香醚（ＢＨＡ）对ＨＢＶ转基因小鼠肝肿瘤的防护作用。方法２６只ＨＢＶ转基因和２４只非转基因小鼠，用生化法测定肝脏醌还原酶（ＱＲ）和谷胱甘肽Ｓ┐转移酶（ＧＳＴｓ）活性、丙二醛（ＭＤＡ）含量和免疫组化法研究肝肿瘤ＰＣＮＡ表达。结果转基因小鼠ＢＨＡ组肝癌发生率０，显著低于普饲组４２％（５／１２例）。ＢＨＡ诱导肝脏ＱＲ和ＧＳＴ活性升高３～７倍，降低ＭＤＡ。ＢＨＡ组肝腺瘤ＰＣＮＡ标记指数３．４９±２．４２％，显著低于普饲组（５．４８±２．２９％）。结论ＢＨＡ长期应用有效延缓了ＨＢＶ转基因小鼠肝肿瘤的发生发展进程。     

重组人血管内皮抑制素对肺微血管内皮细胞增殖及其周期分布的影响

目的：探讨重组人血管内皮抑制素（恩度）对肺微血管内皮细胞增殖的影响及机制。方法：以人非小细胞肺癌细胞系Ａ５４９和人肺微血管内皮细胞系ＨＰＭＥＣ建立非接触式共培养血管模型,并在此模型上应用ＭＴＴ方法研究肿瘤血管内皮细胞的增殖,以流式细胞仪检测血管内皮细胞的周期分布,以ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测血管内皮细胞的ｃｙｃｌｉｎＤ１转录水平和蛋白表达水平的变化。结果：恩度能明显抑制共培养模型下血管内皮细胞的增殖,并呈剂量依赖性;肿瘤内皮细胞Ｇ０／Ｇ１细胞数增多,Ｓ期细胞显著减少;转录和蛋白检测水平ｃｙｃｌｉｎＤ１呈剂量依赖性下调。结论：恩度能抑制肿瘤血管内皮细胞增殖,且这一作用与抑制ｃｙｃｌｉｎＤ１表达,使肿瘤血管内皮细胞滞留于Ｇ０／Ｇ１期有关。     

HBV转基因小鼠的解毒活力变化的研究

ＡＦＢ１和ＨＢＶ是致肝癌的两个主要因素。研究表明ＡＦＢ１与ＨＢＶ有协同致肝癌作用，但其机理不明。方法利用ＨＢＶ转基因小鼠模型，分析小鼠肝组织的细胞色素Ｐ４５０酶的含量和活性，ＧＳＨ和ＧＳＴ的含量和活性，并测定ＨＢＶ转基因小鼠暴露于ＧＳＨ和ＧＳＴ的含量的含量和活性，并测定了ＨＢＶ转基因小鼠暴露于ＡＦＢ１后血清ＡＦＢ１后血清ＡＦＢ１－白蛋白加成物的含量，结果加成物的含量在ＨＢＶ转基因小鼠都较对照小鼠高     

pGPU6/GFP/Neo-hTERT-shRNA对结直肠癌SW480细胞裸鼠移植瘤的抑制

目的:研究靶向hTERT基因的重组质粒pGPU6/GFP/Neo-hTERT-shRNA对人结直肠癌SW480细胞裸鼠移植瘤的治疗作用。方法:于裸鼠右侧腋下皮下注射人结直肠癌SW480细胞建立结直肠癌移植瘤动物模型,随机分为生理盐水组(NS组)、pGPU6/GFP/Neo-NC-shRNA组(NC-shRNA组)和pGPU6/GFP/Neo-hTERT-shRNA组(hTERT-shRNA组),各组连续进行相应治疗6次后,观察肿瘤的生长状况,测量肿瘤体积并绘制肿瘤生长曲线,H-E染色观察肿瘤组织形态学变化,免疫组织化学法检测移植瘤组织中hTERT蛋白的表达,TUNEL法检肿瘤组织中细胞凋亡情况,RT-PCR法检测瘤组织中hTERT mR-NA的表达。结果:与NC-shRNA组和NS组比较,hTERT-shRNA组移植瘤体积增长速度减慢;hTERT-shRNA组移植瘤组织中见肿瘤细胞形态明显改变,凋亡细胞数明显增多[(36.30±5.05)%vs(5.25±1.06)%、(6.95±1.07)%,P<0.01];hTERT-shRNA组hTERT的mRNA和蛋白表达均明显受到抑制(171.42±30.94 vs 146.89±21.43、137.35±25.49,P<0.01;0.39±0.09 vs 0.81±0.335、0.750±0.206,P<0.05)。结论:重组质粒pGPU6/GFP/Neo-hTERT-shRNA通过下调hTERT mRNA和蛋白水平的表达促进肿瘤细胞的凋亡,抑制结直肠癌移植瘤的生长。     

Expression of transgenic carcinoembryonic antigen (CEA) in tumor-prone mice: An animal model for CEA-directed tumor immunotherapy

Carcinoembryonic antigen (CEA) is a tumor marker for the most common forms of adenocarcinomas. We have previously described C57BL/6 mice transgenic for the complete human CEA gene. Compared with humans, these mice reveal a conserved spatiotemporal CEA expression pattern. To establish animal models for CEA-targeted tumor immunotherapy, we have crossed CEA transgenic mice with mice that are genetically predisposed to tumor development. These immunocompetent animals should allow optimization of immunotherapy strategies for maximal destruction of tumor tissues with minimal damage to CEA-expressing normal tissues. To develop a breast tumor model, CEA transgenic mice were cross-bred with mice transgenic for the rat neu protooncogene controlled by the mouse mammary tumor virus long terminal repeat. Female offspring developed poorly differentiated breast tumors, none of which, however, expressed CEA. As a model for colorectal tumors, mice bearing a mutation in the Apc gene (Min mice) and the CEA transgene developed multiple intestinal adenomas with strong CEA expression in all tumor cells. CEA expression had no significant effect on tumor growth. Occasional, well-differentiated breast adenocarcinomas in female offspring expressed CEA focally in tumor cells lining pseudolumina. Cross-breeding Apc^Min/+ mice with neu transgenic mice did not reveal a synergistic effect on the kinetics of breast tumor formation. Finally, CEA transgenic mice crossbred with mice transgenic for the SV40 large T antigen regulated by the surfactant protein-C promoter, developed multiple lung adenocarcinomas that revealed a mosaic CEA expression pattern. Int. J. Cancer 72:197–202, 1997. © 1997 Wiley-Liss Inc.     

异种血管内皮生长因子受体重组蛋白疫苗与顺铂联合给药对小鼠LL/2肿瘤的抑制作用

背景与目的:生物鄄化学治疗是生物治疗与化疗相结合的一种恶性肿瘤综合治疗的新动向之一。抗肿瘤血管形成已成为肿瘤生物治疗的一种策略。本实验观察以重组鹌鹑血管内皮生长因子受体2(quailvascularendothelialgrowthfactorreceptor鄄2,qVEGFR)作为肿瘤疫苗,联合化疗药物顺铂抑制小鼠LL/2肿瘤生长的作用。方法:在C57BL/6小鼠建立LL/2肺癌模型,接种瘤细胞7天后将小鼠随机分成qVEGFR联合顺铂组(简称联合给药组)、qVEGFR组、顺铂组、生理盐水对照组4组。实验中观察肿瘤的生长情况以及小鼠的存活情况和不良反应,并检测小鼠抗自身VEGFR鄄2抗体产生情况、肿瘤微血管密度(microvesseldensity,MVD)、肿瘤细胞凋亡情况等。结果:联合给药组肿瘤明显小于生理盐水对照组,其中有3只小鼠肿瘤完全消退;在接种肿瘤细胞后第70天,联合给药组小鼠存活率为90%,明显高于qVEGFR组(60%)、顺铂组(0%)以及生理盐水对照组(0%)。显微镜下计数结果显示,联合给药组与qVEGFR组所产生的分泌抗qVEGFR抗体的B细胞数量分别为(156.8±19.3)/106个脾细胞和(143.6±18.6)/106个脾细胞。联合给药组肿瘤MVD为11.4±1.3,qVEGFR组为16.4±1.6,顺铂组为33.5±4.5,生理盐水对照组为45.5±4.5,联合给药组MVD明显低于生理盐水对照组。原位末端标记技术     

肿瘤坏死因子相关凋亡诱导配体对非小细胞肺癌细胞凋亡诱导的基础研究

目的:观察肿瘤坏死因子相关凋亡诱导配体对非小细胞肺癌小鼠模型肿瘤细胞凋亡诱导的作用。方法:30只小鼠接种浓度为5×106/ml非小细胞肺癌细胞Tc-1。荷瘤小鼠随机分为6组（空白对照组和5种浓度rhTRAIL实验组）。空白对照组每天注射一次灭菌生理盐水,实验组每天注射浓度不同的rhTRAIL溶液,各组均连续注射15d。每2d测量小鼠肿瘤长短直径,计算肿瘤体积。取小鼠脾脏细胞培养,测量培养液上清TNF-α浓度。结果:rhTRAIL给药组小鼠肿瘤体积明显低于空白对照组（P<0.05）,随着rhTRAIL浓度的增加,肿瘤体积相应减小;小鼠的Tc-1细胞凋亡率随着rhTRAIL浓度增高,其凋亡率逐渐升高;rhTRAIL给药组小鼠的TNF-α浓度均明显高于空白对照组,组间差异具有显著性（P<0.05）。结论:rhTRAIL能有效增强非小细胞肺癌小鼠体内的细胞因子分泌,诱导荷瘤小鼠的肿瘤细胞凋亡,表明rhTRAIL对非小细胞肺癌小鼠的肿瘤细胞具有一定的抑制作用。     

Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background

In human cutaneous malignant melanoma, a predominance of activated mutations in the N-ras gene has been documented. To obtain a mouse model most closely mimicking the human disease, a transgenic mouse line was generated by targeting expression of dominant-active human N-ras (N-RasQ61K) to the melanocyte lineage by tyrosinase regulatory sequences (Tyr::N-RasQ61K). Transgenic mice show hyperpigmented skin and develop cutaneous metastasizing melanoma. Consistent with the tumor suppressor function of the INK4a locus that encodes p16INK4A and p19(ARF), >90% of Tyr::N-RasQ61K INK4a-/- transgenic mice develop melanoma at 6 months. Primary melanoma tumors are melanotic, multifocal, microinvade the epidermis or epithelium of hair follicles, and disseminate as metastases to lymph nodes, lung, and liver. Primary melanoma can be transplanted s.c. in nude mice, and if injected i.v. into NOD/SCID mice colonize the lung. In addition, primary melanomas and metastases contain cells expressing the stem cell marker nestin suggesting a hierarchical structure of the tumors comprised of primitive nestin-expressing precursors and differentiated cells. In conclusion, a novel mouse model with melanotic and metastasizing melanoma was obtained by recapitulating genetic lesions frequently found in human melanoma.     

人血管能抑素抑制小鼠Lewis肺癌移植瘤生长、转移及血管新生

目的:探讨人血管能抑素(canstatin)对小鼠Lewis肺癌移植瘤生长、转移和血管新生的影响.方法:将pCMV-Script/canstatin及空载体pCMV-Script通过电穿孔的方法转染A549细胞,G418筛选获得阳性克隆.RT-PCR检测转染后细胞中canstatin mRNA的表达,Western blotting检测转染后细胞中canstatin蛋白的表达.建立Lewis肺癌小鼠移植瘤模型,观察pCMV-Script/canstatin组A549细胞培养上清对小鼠Lewis肺癌移植瘤的治疗作用,免疫组化检测各治疗组荷瘤小鼠移植瘤的微血管密度.结果:pCMV-Script/canstatin转染A549细胞在G418筛选后成功形成克隆,转染的A549细胞能有效表达canstatin mRNA和蛋白.pCMV - Script/canstatin治疗组小鼠肿瘤体积明显小于pCMV-Script组和NS组[(1.47 ±0.21)cm3 vs(2.43 ±0.15) cm3、(2.53 ±0.18) cm3,P ＜0.01);pCMV-Script/canstatin组、pCMV - Script组和NS组的肺转移结节数分别为(3.00±1.00)、(7.80±1.48)、(7.60 ±2.41)个,pCMV-Script/canstatin组肿瘤转移受到显著的抑制(P＜0.01);pCMV-Script/canstatin组小鼠的肿瘤组织微血管数明显少于pCMV-Script组和NS组[(84.40 ±8.83) vs (188.68 ±11.15)、(190.24±12.91)个,P＜0.01].结论:pCMV-Script/canstatin能在A549细胞中表达并分泌至细胞外,canstatin可明显抑制Lewis肺癌移植瘤的生长、转移和血管新生.     

美洲大蠊提取物对小鼠3LL肺癌的抑制作用及其机制探讨

背景与目的 美洲大蠊系列昆虫药品康复新在体外能诱导多种肿瘤细胞系发生细胞凋亡。本研究旨在探讨美洲大蠊提取物对C57BL／6J小鼠3LL肺癌的抑制效应及其作用机制。方法 荷3LL肺癌的C57BL／6J小鼠随机分为生理盐水组、美洲大蠊提取物高、低剂量组，通过检测各组小鼠的体重变化和抑瘤率，应用流式细胞术进行DNA倍体分析、TUNEL分析和凋亡相关基因表达改变的检测。结果 与生理盐水组比较，美洲大蠊提取物高、低剂量组的抑瘤率分别为41．24％及81．08％。流式细胞术检测美洲大蠊提取物能诱导肿瘤细胞凋亡，随着药物剂量的增高，其凋亡率也升高，S期及G2／M期细胞数大量减少，细胞被阻滞在G0／G1期；流式细胞术TUNEI。检测结果显示，细胞凋亡和坏死同时存在。流式细胞法分析3LL肺癌细胞凋亡相关基因显示，无论是低剂量组还是高剂量组，美洲大蠊提取物均能上调Fas、FasR、p53等凋亡基因表达，下调Bcl-2抑制凋亡基因表达。结论 美洲大蠊提取物对3LL肺癌生长具有一定的抑制作用，能促进肿瘤细胞凋亡，其机制可能与调控细胞凋亡相关基因的表达有关。     

Delays in malignant tumor development in transgenic mice by forced epidermal keratin 10 expression in mouse skin carcinomas

The keratin cytoskeleton is formed in different epidermal compartments by distinct polypeptides. Basal, proliferative keratinocytes express keratin (K) 5 and K14, whereas, suprabasal, post-mitotic keratinocytes express K1 and K10. Changes in this keratin pattern have been found to occur in hyperproliferative skin disorders and, in particular, throughout mouse epidermal carcinogenesis. Whereas some keratins not found in normal epidermis (K6, K16, K13, and K8) are induced at different stages of tumor development, K1 and K10 expression is lost. To determine whether K1 and K10 loss is just a consequence of the altered differentiation program or an event required for tumor progression, we generated transgenic mice carrying the human keratin 10 gene (hK10) under the control of a bovine keratin 6 gene regulatory region, which is silent in normal skin but is induced and drives transgene expression in hyperproliferative skin keratinocytes and, therefore, in skin tumors. Transgenic animals subjected to a complete carcinogenesis protocol developed tumors that contained various amounts of transgenic hK10. Although no significant difference was found in tumor number or malignancy, tumor onset was significantly delayed in transgenic mice, indicating that the presence of K10 actually impairs tumor development. Mol. Carcinog. 20:3–9, 1997. © 1997 Wiley-Liss, Inc.     

Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A₁₆₅ (hVEGF-A₁₆₅) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A₁₆₅ in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A₁₆₅ mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A₁₆₅ overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A₁₆₅, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A₁₆₅ in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A₁₆₅-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A₁₆₅ transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.     

红色荧光蛋白和荧光素酶双报告基因转基因小鼠的建立

背景与目的:活体动物体内光学成像(optical in vivo imaging)主要采用生物发光与荧光两种技术。生物发光是用荧光素酶(luciferase,Luc)基因标记细胞或DNA,而荧光技术则采用荧光报告基团(GFP、RFP、Cyt及dyes等)进行标记,利用一套非常灵敏的光学检测仪器,能够直接监控活体生物体内的细胞活动和基因行为,生物发光成像具有高的灵敏度和特异性,同时生物发光信号可用于精确定量,而荧光成像具有方便、便宜、直观、标记靶点多样和易于被大多数研究人员接受的优点。本研究基于慢病毒介导的转基因方法制备红色荧光蛋白(red fluorescent protein,RFP)和Luc双报告基因转基因小鼠(即RL转基因小鼠),将这两种技术融为一体。方法:制备携带RFP和Luc基因(简写RL基因)的慢病毒,然后将携带RL基因的慢病毒注入小鼠单细胞受精卵卵周隙以感染受精卵,胚胎移植进假孕母鼠以获得仔鼠,应用小动物活体成像仪、体视荧光显微镜和PCR等在蛋白和DNA水平上筛选和鉴定,并获得RL转基因小鼠。结果:移植卵周隙注射有慢病毒的胚胎125枚给6只假孕母鼠,其中4只假孕母鼠怀孕,共生仔鼠20只;利用小动物活体成像仪检测RFP和Luc表达,在蛋白水平证实20只F0代中,3只高表达RFP和Luc;DNA水平检测证实,3只RFP和Luc阳性的小鼠基因组中确实整合有外源转基因RL,预示基因型鉴定结果很好验证了小动物活体成像仪筛选和鉴定结果。此外,RL转基因首建鼠基因组中整合的RL转基因可稳定遗传至下一代,并能正常表达。RL转基因小鼠主要脏器均可见红色荧光和Luc信号,但不同脏器间荧光和Luc强度有差异。结论:成功制备RL双报告基因转基因小鼠,为后续研究干细胞在肿瘤发生、发展和转移中的作用和造血重构等提供双报告基因标记的各种移植用供体细胞,并对此供体细胞及其在体内衍生的细胞进行灵敏的非损伤、实时可视化体内跟踪。