p53基因对移植心脏冠状动脉内膜增厚的抑制

背景:研究表明野生型p53基因具有抑制血管平滑肌细胞增殖、减轻血管内膜损伤后内膜增生和管腔狭窄的作用。目的:观察野生型p53基因对心脏移植后移植心脏冠状动脉内膜增厚的抑制作用。方法:以Wistar大鼠为供体,SD大鼠为受体建立大鼠腹腔异位心脏移植模型,取出供心后,经供心冠状动脉分别注射携带野生型p53基因的重组腺病毒液(Ad-p53组)、携带β-半乳糖酐酶基因的重组腺病毒液(Ad-LacZ组)和生理盐水(对照组)800μL,4℃静置30min后进行心脏移植。移植后5d,采用RT-PCR扩增法检测各组供心冠状动脉组织外源性p53表达;移植后28d,组织学观察移植心脏,计算各组冠状动脉内膜与中膜厚度比和血管壁厚度与管腔直径比。结果与结论:移植后5d,Ad-p53组供心内可见野生型p53基因的表达。移植后28d,Ad-p53基因组冠状动脉内膜与中膜厚度比和血管壁厚度与管腔直径比均较Ad-LacZ组及对照组明显减小(P0.05),Ad-LacZ组和对照组比较差异无显著性意义(P0.05)。结果证实在心脏移植中,采用冠状动脉注射法可以将目的基因转移至供心内,供心内转染野生型p53基因能显著抑制移植心脏冠状动脉内膜增生,减轻管腔狭窄程度。     

小鼠心脏移植保存期间经体外灌注行重组腺病毒基因转染的研究

目的:探讨小鼠心脏移植中,通过在体外保存期间对供心进行灌注,进行重组腺病毒基因转染的方法及效率。方法: 利用小鼠异位心脏移植,在供心保存期间行重组腺病毒灌注,在移植后7 d检测移植物内标记基因的表达情况,并通过免疫组织化学染色检测移植物内免疫细胞的浸润,通过流式细胞仪检测受体淋巴细胞的激活状态。结果: 小鼠供心通过体外灌注进行重组腺病毒基因转导,并在4 ℃保存2 h,复跳率达100%,移植后1周,标记基因表达强度到达峰值。移植物组织结构完整,无明显细胞浸润。受体淋巴细胞的激活状态与空白对照组相比,无明显差异。结论: 小鼠心脏移植保存期间体外灌注可应用于腺病毒基因转染等移植物处理过程。     

腺病毒介导的CD40Ig基因治疗诱导异基因大鼠心脏移植物的长期存活

目的:通过腺病毒介导的CD40Ig基因治疗,阻断受体体内CD40/CD154共刺激途径,诱导异基因大鼠之间的心脏移植耐受,并探讨相关机制。在将异基因DA大鼠的心脏移植给受体LEW大鼠后,即经门静脉输注携带CD40Ig基因的腺病毒(AdCD40Ig,5×109 pfu)。观察、记录心脏移植物的存活时间。ELISA检测受体大鼠体内CD40Ig蛋白的表达。通过MLR,IL-2逆转实验及Th1/Th2型细胞因子表达的检测,研究耐受的机制。结果:与未处理对照组相比,携带绿色荧光蛋白基因的腺病毒(AdEGFP)给予组,移植物的存活时间未见明显延长;AdCD40Ig腺病毒给予组,异基因大鼠心脏移植物平均存活时间延长至(142.8±26.8)天。ELISA检测表明,CD40Ig蛋白在受体体内表达时间较长,但随时间的推移表达水平逐渐下降。Th1和Th2型细胞因子的检测结果显示,耐受大鼠体内未发现这两类细胞因子偏移的现象。MLR证明耐受大鼠对供体抗原表现为特异性低应答。IL-2逆转实验表明,该耐受形成的早期与克隆无能有关。结论:经.AdCD40Ig腺病毒基因治疗的受体大鼠可以产生特异性的移植耐受,使异基因大鼠心脏移植物长期存活。     

外源性野生型p53基因在两个肺腺癌细胞系中表达及对p16和p21基因的调控

目的 探讨p53基因在肺癌细胞周期中的作用机制，以及裸鼠体内基因治疗的作用。方法 用以腺病毒为载体的野生型p53基因pAdCMV-p53(Ad-p53)感染高转移肺腺癌95D细胞系和高浸润肺腺癌L-18系，对感染前后各细胞系的细胞生长曲线、p53、p16和p21基因的表达以及调亡进行分析。此外，用Ad-p53对两细胞系进行了裸鼠体内感染实验。结果 体外实验中，导入p53基因细胞系的生长均得到抑制，并且最终都出现凋亡，感染后的细胞中p53和p21基因的mRNA表达量明显增高，p16基因的mRNA表达量则无明显变化。p53基因治疗后的裸鼠，其中95D细胞系的肿瘤全部消失；L-18细胞系的腹腔注射组肿瘤全部消失，而皮下注射组无明显变化。结论 p53基因是一个有效的肿瘤抑制基因，其诱导细胞凋亡的途径与p16基因不同。腺病毒介导的野生型p53基因可以有效地控制肺癌细胞的发展和转移。     

野生型p53基因导入对U937细胞分化、凋亡和CD36受体表达的影响

目的： 研究野生型p53基因重组腺病毒载体（AdCMV-p53）导入对U937细胞分化、凋亡和清道夫受体CD36表达的影响。 方法： AdCMV-p53导入U937细胞后，用细胞计数、细胞周期分析、台盼蓝染色排除法计数细胞悬液中的活细胞数目和NBT还原反应观察其对U937细胞生长、分化的影响；RT-PCR、免疫荧光和流式细胞分析检测AdCMV-p53导入对CD36表达的影响。 结果： AdCMV-p53可以高效导入U937细胞，野生型p53基因导入促进U937细胞向巨噬细胞分化，台盼蓝染色发现实验组阳性细胞数（64.6±9.2）%较对照组（14.2±5.5）%明显增多，吞噬能力增强；NBT还原反应实验组（49.7±12.6）%较对照组（6.3±1.8）%升高。RT-PCR和流式细胞分析检测，野生型p53基因导入使得CD36 mRNA转录增强，CD36蛋白表达增加。 结论： 野生型p53基因能影响细胞分化和凋亡，并上调清道夫受体CD36的表达，对于动脉粥样硬化的预防和基因治疗具有潜在意义。     

供体与受体移植前预处理延长大鼠移植心脏成活时间

目的：用供体品系大鼠皮肤致敏方法及腹腔内异位心脏移植模型研究雷公藤多式供体预处理及供体脾细胞受体胸腹内注射诱导免疫耐受对移植心脏存活影响及二种方法的协同效果。方法：SD大鼠作供体,Wistar大鼠作受体,所有受体在心脏移植前均无用供体皮肤致敏,A组作单纯心脏移植,B组作经供体预处理后的供心移植,C组用供体脾细胞胸腺内注射诱导耐受后作心脏移植,D组考察供受体两种预处理方法的协同效果。结果：各处理组供心存活时间均校对照组明显延长,C,D二组病检见淋巴细胞浸润及心肌坏死明显减轻,但微血管病变较严重。结论：两种方法均能明显延长移植供心成活时间,并有良好的协同效果,经胸腺途径诱导的耐受能明显抑制细胞免疫,但对急性血管排斥没有预防作用。     

а-MSH基因转染的小鼠同种异体移植心脏存活期延长

目的 观察a-黑素细胞刺激素(a-MSH)基因原位转染供者小鼠心脏对小鼠同种异体心脏移植排斥反应的影响.方法 将携带a-MSH基因和绿色荧光蛋白(GFP)基因的重组腺相关病毒(rAAV-a-MSH)和单纯携带GFP的重组腺相关病毒(rAAV-GFP)分别经主动脉灌注原位转染供者小鼠心脏,再行同种异体心脏移植,观察心脏移植物的存活时间.测定移植心脏的GMSH表达情况,并于术后7 d测定移植心脏的细胞因子及趋化因子水平.结果 腺相关病毒载体能有效地将a-MSH基因导人小鼠心脏,与rAAV-GFP空载体组[(7.0±0.33)d]和PBS组[(7.0±2.23)d]相比,rAAV.a-MSH原位转染组[(16.3±2.21)d]的心脏移植物存活时间延长.术后7 d,rAAV-a-MSH原位转染组心脏移植物的Thl细胞因子(IL-2、IFN-a)水平较其它两组减低,而Th2细胞因子(IL4、IL-10)水平升高,趋化因子MCP-I、RANTES水平下降.结论 携带a-MSH基因的rAAV载体经原位转染供者小鼠心脏,能促进移植心脏局部的细胞因子格局由Th1型向Th2型偏移,降低趋化因子水平,从而延长同种异基因移植心脏的存活时间.     

重组腺病毒介导的p16INK4a表达诱导A549细胞衰老的研究

目的 构建E1区缺失的复制缺陷型5型重组腺病毒载体，探讨p16INK4a基因对肺癌细胞株A549细胞增殖与衰老的影响。方法 通过脂质体介导，将pAdCMV-p16INK4a与pJM17共转染人5型腺病毒E1基因转化的人胚肾细胞系293细胞，同源重组产生腺病毒空斑，用双重PCR筛选出携带p16INK4a基因的重组腺病毒并感染肺癌细胞A549，用免疫组化及Western blot检测腺病毒载体介导的基因转移效率和蛋白表达水平，分别用X-gal染色和TRAP-ELISA检测A549细胞中衰老相关β-半乳糖苷酶及端粒酶活性。结果 腺病毒载体可将95％以上的p16INK4a基因转移到A549中，受感染细胞有外源p16INK4a蛋白表达，其生长受到明显抑制，即p16INK4a基因能诱导A549细胞表达衰老相关β-半乳糖苷酶，并抑制细胞中的端粒酶活性。结论 复制缺陷型重组腺病毒，能有效地介导外源基因的转移与表达，可用于基因免疫和基因治疗；p16INK4a基因能抑制肺癌细胞A549生长并诱导其发生复制性衰老。     

大鼠过氧化物酶体增生因子活化受体α基因重组腺病毒载体及在乳鼠心肌细胞中的表达

背景：过氧化物酶体增生因子活化受体α是调节心脏脂肪酸氧化的重要核转录因子,利用基因工程技术构建其重组腺病毒载体对研究心脏能量代谢障碍机制具有重要意义。 目的：构建大鼠过氧化物酶体增生因子活化受体α基因重组腺病毒载体,评价其在原代培养的乳鼠心肌细胞中的表达效率。 方法：采用RT-PCR法自SD大鼠肝脏扩增过氧化物酶体增生因子活化受体α基因,将其克隆至带有增强型绿色荧光蛋白的穿梭质粒载体pAd-Track-CMV。线性化重组穿梭质粒载体并转化入含有腺病毒骨架质粒pAd-Easy-1的感受态大肠杆菌BJ5183构建重组腺病毒载体质粒。用脂质体将线性化的重组腺病毒载体质粒转染于人胚肾293细胞以包装、扩增,纯化病毒后计算滴度。用纯化的重组腺病毒转染心肌细胞,荧光显微镜观察转染效率;荧光定量PCR、免疫印迹法检测细胞过氧化物酶体增生因子活化受体α mRNA、蛋白表达。 结果与结论：成功构建携带大鼠过氧化物酶体增生因子活化受体α基因的重组腺病毒载体,病毒滴度为3.5×10¹¹ pfu/L。重组腺病毒转染心肌细胞的效率达90%以上,被转染细胞目的基因mRNA、蛋白表达显著增高。该载体的成功构建为研究过氧化物酶体增生因子活化受体α基因在心肌能量代谢障碍中的作用奠定基础。     

大鼠异位心脏移植模型的改进及组织学研究

目的:在Ono模型基础上,建立简单、可靠的大鼠腹腔异位心脏移植模型。方法:以Wister大鼠为供体,SD大鼠为受体,改良建立大鼠腹腔异位心脏移植模型,分为3组:正常Wister大鼠作为对照组;移植后亚急性排斥反应组(术后2周);移植后慢性排斥反应组(术后8周)。分组喂养后按规定时间收获心脏标本进行组织形态学观察。结果:手术成功率为87.5%。其中麻醉过深死亡2例,疑青霉素过敏死亡1例,术中出血过多死亡4例。供心移植吻合时间(24.3±7.6)min,供心冷缺血时间(34.5±8.2)min,腹部触诊移植心脏搏动时间(16.8±7.4)d。组织形态学观察示:移植心脏心肌组织内有大量粒细胞浸润;随着移植后时间延长,CAV逐渐加重,冠脉数量减少,心肌萎缩并心肌纤维化发生。结论:改良Ono模型建立的心脏移植模型具有简单、可靠的优点。收获的移植心脏标本在组织形态学上反映了CAV和心肌纤维化发生、发展的过程,为研究心脏移植后相关疾病提供理想的组织标本。     

Time-dependent expression of ICAM-1 & VCAM-1 on coronaries of the heterotopically transplanted mouse heart.

To investigate the pathogenesis of accelerated graft atherosclerosis after cardiac transplantation, a genetically well-defined and reproducible animal model is required. We performed heterotopic intraabdominal heart transplantation between the two inbred strains of mice. Forty hearts from B10.A mice were transplanted into B10.BR mice. Recipients were sacrificed at 1, 3, 5, 7, 14, 28, and 42 days after implantation. The specimens from both donor and recipient were examined with fluorescent immunohistochemistry and the serial histopathologic changes were evaluated. In the donor hearts, ICAM-1 and VCAM-1 expressions were minimal at day 1 and they gradually increased, reaching their peaks on day 5 or 7 and remained unchanged by day 42. However, there were very little expressions in the recipients' hearts. Mean percent areas of intima in the donor coronaries revealed progressive increase by day 42. However, those in the recipients occupied consistently less than 5% of the lumen. In conclusion, we demonstrated that a heterotopic murine heart transplantation model was a useful tool to produce transplantation coronary artery disease and that adhesion molecules on the cardiac allografts were activated very early and remained elevated at all time-points, nonetheless the arterial lesion was detected after day 28 and its progression was accelerated thereafter.     

Adenoviral-mediated transfer of p53 gene enhances TRAIL-induced apoptosis in human hepatocellular carcinoma cells

p53 is a tumor suppressor protein with numerous biological functions including transformation, regulation of cell growth, differentiation and apoptosis. The TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. We investigated the effects of combining wild-type p53 gene transduction by adenoviral infection (Ad-p53) with addition of TRAIL on cell death, expression levels of TRAIL receptors (TRAIL-R1, TRAIL-R2), FLICE inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) on human hepatocellular carcinoma (HCC) cell lines. HCC cell death was increased by combination of Ad-p53 infection and addition of TRAIL compared to either alone. Western blotting demonstrated decreased TRAIL-R1 and TRAIL-R2 levels after infection with Ad-p53. FLIP levels decreased in Huh7 cells and Hep3B cells, and XIAP levels decreased in all three HCC cell lines after infection with Ad-p53. Thus, death of HCC cells due to combined p53 gene transduction and exogenous TRAIL may be due to down regulation of FLIP or XIAP.     

Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts.

In heart transplantation, long-term engraftment success is severely limited by the rapid development of obliterative disease of the coronary arteries. Data from various groups have been suggestive of a pathogenetic role of herpesviruses, particularly human cytomegalovirus, in accelerated allograft coronary artery disease; however, results are not yet conclusive. This study examines the hypothesis that human cytomegalovirus infection of allograft tissues is related pathogenetically and directly to accelerated coronary artery disease. Using in situ DNA hybridization and polymerase chain reaction, we examined particular coronary artery segments from 41 human heart allografts (ranging from 4 days to greater than 4 years after transplantation; mean, 457 days) and 22 donor age- and gender-comparable, coronary site-matched trauma victims for presence of human cytomegalovirus DNA. Human cytomegalovirus genome was detected in 8 of 41 (19.5%) allografts and in 1 of 22 (4.5%) control hearts. This difference in positivity was not statistically significant (P = 0.10). In the human cytomegalovirus-positive hearts, viral genome was localized to perivascular myocardium or coronary artery media or adventitia. Human cytomegalovirus genome was not detected in arterial intima of any allograft or control heart, although human cytomegalovirus genome was readily identified within intima of small pulmonary arteries from lung tissue with human cytomegalovirus pneumonitis. By statistical analyses, the presence of human cytomegalovirus genome was not associated with the nature or digitized extent of transplant arteriopathy, evidence of rejection, allograft recipient or donor serological data suggestive of human cytomegalovirus infection, duration of allograft implantation, or causes of death or retransplantation. Thus, our data indicate a low frequency of detectable human cytomegalovirus genome in accelerated coronary artery disease and do not support a direct role for human cytomegalovirus in vascular wall infection or in the development of accelerated coronary artery disease.     

血管内皮生长因子基因转染骨髓间充质干细胞同种异体移植治疗大鼠急性心肌梗死

目的探讨同种异体血管内皮生长因子(VEGF)基因转染的骨髓间充质干细胞(MSCs)在大鼠梗死心脏局部存活、分化及对心功能的影响;明确同种异体干细胞及VEGF基因转染干细胞移植治疗急性心肌梗死(AMI)的可行性及效果。方法雄性SD大鼠30只,随机分为单纯注射培养基对照组、MSCs治疗组及VEGF基因转染MSCs治疗组。分离纯化雄性Wistar大鼠骨髓间充质干细胞(rMSCs),于左冠状动脉前降支结扎1h后植入到SD大鼠心组织,移植4周后检测心功能并取心脏行组织染色检查。结果异体大鼠MSCs可在梗死心组织定居、生存;免疫组化检测MSCs转化为心肌细胞及血管内皮细胞;与对照组比较VEGF基因转染异体细胞移植组左室射血分数升高(P<0.05),梗死边缘区心肌面毛细血管数目明显增加(P<0.05)。结论同种异体VEGF基因转染MSCs移植治疗AMI可行、有效。     

大鼠心脏移植后淋巴细胞5种免疫分子表达的变化

目的：观察大鼠异体异位心脏移植术后不同时间点，淋巴细胞相关免疫分子的表达水平、供心存活率及心肌组织的病理学改变，探讨免疫排斥反应的相关时间进程。方法：分别经供体SD大鼠的心脏主动脉、肺动脉与受体Wistar大鼠的腹主动脉及下腔静脉吻合，进行异位心脏移植术。于术前及术后不同时间点，取血分离淋巴细胞。用流式细胞仪分别检测淋巴细胞上CD4、CD8、IL-2R、ICAM-Ⅰ和MHC-Ⅱ类分子的表达水平。对供心心肌组织进行常规病理学检查。结果：于移植后24h，只有MHC-Ⅱ类分子的表达水平增加，CD4、IL-2R和ICAM—Ⅰ的表达水平降低，CD8无改变。术后72h，CD4、CD8及IL．2R的表达增加，其中CD8和IL-2R达峰值。术后7～10d，除CD4的表达继续增加外，其他4种免疫分子的表达水平逐渐降低。术后不同时间点移植心脏的存活率分别为：100％(24h)、85．7％(72h)、16．7％(7d)、0(10d和12d)。病理学检查显示，移植后24h，心肌组织无明显的病理学变化，术后3d，7只大鼠中4只出现ⅠA级及以上病理改变。术后7d，6只大鼠全部发生Ⅱ级以上的病理学变化。结论：大鼠心脏移植后的24h内，外周血淋巴细胞为以MHC-Ⅱ分子表达为主的抗原识别、呈递期，并伴有一过性免疫功能降低；术后24—72h为T细胞活化期，术后3-7d为免疫排斥反应效应期。CD4、CD8和IL-2R等免疫分子表达的峰值与开始出现轻度排斥反应时病理学变化的时间相一致。     

常温不停跳保存供心的超微结构和动力学变化

背景：心脏移植供心保护方法有向心脏正常生理状态改进的趋势,以期延长供心保存时间,改善供心的保存质量。理论上供心常温不停跳灌注保存是一种最接近生理状态的保存方法,有良好的发展潜力。 目的：观察连续灌注不停跳供心常温长时间保存心肌超微结构和左室血流动力学的变化。 方法：广西巴马小型猪24只,按随机数字表法分为不停跳连续灌注组和冷保存组,每组各6对。不停跳连续灌注组供心在顺行连续灌注跳动状态下切取,应用氧合血37 ℃、保存8 h后移植。冷保存组供心应用UW停搏液灌停后,0-4 ℃UW液保存8 h后移植。移植主动脉开放3 h观察供心左心室收缩压、左心室舒张压、左室平均压、左室舒张末压、左室内压最大上升和下降速率。记录移植后心律变化,需要除颤例数,以及主动脉开放3 h后能否脱离体外循环。主动脉开放3 h后取供心左心室前外侧壁心肌组织,观察线粒体及肌膜改变。 结果与结论：不停跳连续灌注组供心移植后左心室收缩压、左室平均压、左室舒张末压、左室内压最大上升和下降速率均优于冷保存组(P < 0.05);主动脉开放后不停跳连续灌注组均可保持窦性心律,冷保存组仅1例自主恢复窦性心律;主动脉开放后3 h不停跳连续灌注组5例可顺利脱机,冷保存组1例可以脱机;不停跳连续灌注组心肌超微结构保存良好。提示供心常温灌注不停跳方法有良好的心肌保护效果,适于供心的长期保存。     

同期心脏和肺移植6例

背景：心肺移植目前仍然是治疗终末期心肺疾病的最好方法,但由于诸多原因国内的供者短缺是一个很严峻的问题。 目的：观察利用同一供者对不同受者同期进行心、肺移植的可行性。 方法：将3例供者的心脏、肺脏分别同期移植给3例相同血型的终末期心脏疾病受者和3例终末期肺脏疾病受者,观察移植效果。 结果与结论：6例患者中有1例双肺移植患者出现右肺上叶静脉栓塞于术后第9天再次手术切除后痊愈,术后30 d出院。1例双肺移植患者1个月出现感染经对症治疗后好转,于术后2个月出院。1例心脏移植患者出现早期肾功能衰竭,经血液透析治疗后痊愈出院。余3例患者均安全渡过围手术期后痊愈出院,到目前有3例仍有很好的生活质量。提示利用同一供者的心、肺分别给不同受者进行心、肺移植,能充分利用供者,缩短受者等待时间。     

Donor brain death leads to differential immune activation in solid organs but does not accelerate ischaemia–reperfusion injury

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia–reperfusion injury (IRI) post‐transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3⁺CD4⁺, CD3⁺CD8⁺ T cells and NKp46⁺ NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post‐transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham‐transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

人突变p27基因对大肠癌细胞生物学行为的影响

目的： 观察人突变p27基因（p27mt）对人大肠癌细胞生长的影响，探讨p27mt基因在大肠癌基因治疗中的作用及可能机制。方法： 以携带突变p27基因复制缺陷型腺病毒（Ad-p27mt）为载体，转染大肠癌细胞SW480；Western blotting方法检测p27mt蛋白的表达；细胞计数法检测p27mt对SW480细胞生长的抑制作用；用流式细胞仪检测细胞周期；用DNA片段分析法检测细胞凋亡。结果： Ad-p27mt转染SW480细胞后，p27在细胞中出现了蛋白高表达;77.96%的细胞阻滞于G₀/G₁期，而Ad-LacZ组及空白对照组分别为27.57%和25.29%；生长曲线显示Ad-p27mt对细胞生长有明显的抑制作用；DNA片段分析示p27mt基因可诱导细胞的凋亡。结论： p27mt对细胞周期有明显的阻滞作用，主要阻滞于G₀/G₁期；p27mt基因对细胞的生长抑制机制与诱导细胞凋亡和细胞周期的阻滞有关。     

Engineering ex vivo-expanded marrow stromal cells to secrete calcitonin gene-related peptide using adenoviral vector

Calcitonin gene-related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated CGRP gene transfer into ex vivo-expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro-CGRP and AdntlacZ, adenoviral vectors containing prepro-CGRP or nuclear-targeted beta-galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral-mediated gene transfer into ex vivo-expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro- CGRP-transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro-CGRP-transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 +/- 0.4 pmol CGRP/1 x 10(6) cells/48 hours (mean +/- standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro-CGRP-transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication-deficient recombinant adenovirus can be used to gene engineer ex vivo-expanded MSCs and that high-level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell-based cell and gene therapy strategy for the treatment of cardiovascular diseases.