Inhibition of Metastatic Progression of SSTR2 Gene Transfection Mediated by Adenovirus in Human Pancreatic Carcinoma Cells

The inhibition of metastatic progression of Somatostatin receptor type 2 （SSTR2） gene transfection mediated by adenovirus in human pancreatic carcinoma cells and the mechanisms involved in this effect were studied. The full-length human SSTR2 cDNA was introduced into the pancreatic cancer cell line BXPC-3 by adenovirus-mediated transfection. Stable expression of mRNAs and protein of SSTR2 was detected by RT-PCR and Western-blot. The Matrigel-coated Transwell was used to detect the migratory and invasive ability of SSTR2-expressing cells, Adv-GFP control cells and mock control cells. Furthermore, the expression of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor of metalloproteinase-2 （TIMP-2） was detected by RT-PCR in these cells. The stable expression of SSTR2 was detected in BXPC-3 transfected by Adv-GFP-SSTR2. A dramatic decrease of BXPC-3 expressing sst2 cells migrating through a Matrigel-coated filter was observed, as compared with Adv-GFP control and mock control cells （P〈0. 01）. Moreover, the expression of MMP-2 mRNA was significantly reduced in the SSTR2-expressing cells and converse- ly the expression of TIMP-2 mRNA was significantly increased in the SSTR2-expressing cells when compared with the Adv-GFP control and mock control （P〈0. 01）. The expression of reintroduced human SSTR2 gene in BXPC-3 cells by Adv-GFP-SSTR2 had the anti-migratory and anti-invasive effects, and the mechanisms involved in this effect may be due to the down-regulated expression of MMP-2 and up-regulated expression of TIMP-2.

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

表达外源基因SPAM1重组CAV-2溶瘤病毒的构建与拯救

旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统构建携带SPAM1外源基因的中间载体,使用中间载体将CAV-2骨架载体E3区域缺失并替换SPAM1外源基因表达盒。再利用合成引物敲除kmccdB反向筛选表达盒构建P15A-CAV-2-mCMV-SPAM1-SV40 polyA感染性克隆质粒并在MDCK-E1A细胞系中拯救重组溶瘤病毒,对重组病毒体外溶瘤效应进行验证。根据测序与酶切验证结果证明,本研究成功构建并拯救出稳定表达外源基因SPAM1的重组溶瘤病毒1株;IFA试验证明,重组毒株能够高水平稳定表达外源基因且外源蛋白具有可弥散分布于细胞间的特点。缺失E3区域表达外源基因SPAM1的重组溶瘤病毒通过光镜观察和CCK8检测发现具有对犬癌细胞系A72强烈的杀伤效应。综上,本研究成功构建1株具有良好溶瘤效应的重组CAV-2,为后续应用于宠物肿瘤治疗奠定了基础。

Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase

3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human （h） and rat （r）. Recombinant Ad-r（h）SYN1-DHCR24 was transfected into AD-293, N2A （mouse neuroblastoma）, and MIN6 （mouse pancreatic carcinoma） cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer＇s disease.

Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2antisense RNA, and to explore its effects on liver cancer cell proliferation.METHODS: We studied the expression of COX-2 in 34cases of hepatocellular carcinoma (HCC) and SMMC7402and SMMC7721 by immunohistochemical technique.Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry.Cell proliferation was determined by colony-forming efficiency.RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06× 1012 PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line.The difference of apoptotic index between the Ad-AShcox2 group and control group was statistically significant(tcontrol group= 32.62 and tAd-LacZ= 10.93, P＜0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and(33.6±4.24)%, respectively.CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.

COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES

Recombinant adenoviruses encoding murine IL 2 gene or IL 3 gene were directly injected into established subcutaneous tumor model of G422 glioblastoma cells. After treatment, the tumor size and survival of the glioblastoma bearing mice were observed. The splenic NK and CTL cytotoxicities were detected by standard 4 hour 51 Cr release assay. We also examined the histopathological changes of tumor by hematoxylin and eosin staining. The results showed that intratumoral injection of adenoviruses encoding murine IL 2 gene or IL 3 gene significantly inhibited the growth of G422 glioblastoma and prolonged the survival period of glioblastoma bearing mice. The CTL cytotoxicity of the gene therapy groups was significantly higher than that of the control groups, but NK activity remained unchanged, indicating that specific immunity contributes to the in vivo antitumor effect of the direct gene therapy. There were much more tumor necrosis and inflammatory cell infiltration in the tumor of the gene therapy groups. Combined IL 2/IL 3 gene therapy could induce higher level of CTL and enhance the therapeutic potential further. The results suggest that intratumoral injection of recombinant adenoviruses encoding certain kind of cytokines may be a useful approach in the treatment of a malignancy of the central nervous system.

Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

Objective:To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro.Methods:NDRG2 was harvested by RT-PCR,confirmed by DNA sequencing,and then cloned into the eukaryotic expression vector pIRES2-EGFP,which encodes green fluorescent protein(GFP),to construct p1RES2-EGFP-NDRG2 plasmid.OS-RC-2 cells with NDRG2 negative expression were transfected with p1RES2-EGFP-NDRG2 plasmid.The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes.After colony-forming cell assays,cell proliferation detection and MTT assays,the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells.Cell cycle was determined by flow cytometry.Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle.Results:A eukaryotic expression vector p1RES2-EGFP-NDRG2 was successfully constructed.After NDRG2 transfection,the growth of OS-RC-2 cells was inhibited.Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present,and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion.Conclusions:NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

Optical control after transfection of channelrhodopsin-2 recombinant adenovirus in visual cortical cells

Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Long Evans rats at postnatal day 1 were used for primary culture of visual cortical cells and 24 hours later, cells were transfected with recombinant adenovirus carrying channelrhodopsin-2 and green fluorescent protein genes. After 2 4 days of transfection, green fluorescence was visible in the cultured cells. Cells were stimulated with blue light （470 nm）, and light-induced action potentials were recorded in patch-clamp experiments. Our findings indicate that channelrhodopsin-2-recombinant adenovirus transfection of primary cultured visual cortical cells can control the production of action potentials via blue light stimulation.

犬腺病毒2型的分离鉴定和致病性分析

为了解湖北省犬腺病毒2型(CAV-2)的基因遗传变异特征,本试验从武汉市某比格犬养殖场采集免疫过犬四联活疫苗的临床发病犬的眼、鼻和肛拭子,将经PCR检测为CAV-2阳性的病料接种犬肾细胞(MDCK细胞)进行病毒分离,对CAV-2分离株进行电子显微镜观察和间接免疫荧光鉴定,E3、Fiber、Penton和E1b基因PCR扩增和测序分析以及致病性试验。结果显示:经分离和鉴定共获得3株CAV-2,分别命名为HB404株、HB405株和HB078株。3株CAV-2分离株的E3、Fiber、Penton和E1b基因与国外疫苗毒株相似性分别为98.4%~99.6%、99.3%~99.9%、99.8%~99.9%和99.5%~99.7%;3株分离株的E3、Fiber和Penton基因与国内流行毒株相似性分别为96.6%~97.5%、98.5%~99.7%和99.4%~99.9%。且3株CAV-2分离株与国外疫苗毒株在同一分支,与国内流行毒株不在同一分支。HB405株能使犬出现精神不振、食欲减退、眼鼻有分泌物、打喷嚏和咳嗽等临床症状。结果表明,3株分离株的亲缘关系与国外疫苗毒株较近,HB405株为强毒株,能够使犬发病。本试验结果可为CAV-2的诊断和预防提供参考依据。

缺氧诱导因子1α、Bcl-2/腺病毒E1B19kDa相互作用蛋白3在儿童创伤性脑损伤中的表达及意义

目的动态观察缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)、Bcl-2/腺病毒E1B19kDa相互作用蛋白3(Bcl-2/adenovirus E1B19kDa-interacting protein 3,BNIP3)在儿童创伤性脑损伤(traumatic brain injury,TBI)中的变化,并评估其预测儿童TBI病情轻重及预后的临床价值。方法前瞻性纳入2021年1月—2023年7月47例中重度TBI患儿为研究对象,根据格拉斯哥昏迷评分分为中度亚组(9~12分)和重度亚组(3~8分);以同期因腹股沟斜疝诊治且无基础疾病的30例患儿为对照组。比较各组HIF-1α、BNIP3、自噬相关蛋白Beclin-1及S100B水平的差异,采用受试者操作特征曲线(receiver operating characteristic curve,ROC曲线)评估HIF-1α、BNIP3、Beclin-1及S100B对TBI病情轻重及预后的预测价值。结果TBI组患儿血清HIF-1α、BNIP3、Beclin-1、S100B水平高于对照组(P<0.05);TBI患儿中,重度亚组HIF-1α、BNIP3、Beclin-1、S100B水平高于中度亚组(P<0.05)。相关性分析显示,血清HIF-1α、BNIP3、Beclin-1、S100B水平与格拉斯哥昏迷评分呈负相关(P<0.05)。治疗7 d后,非手术TBI和手术TBI患儿的血清HIF-1α、BNIP3、Beclin-1及S100B水平均较治疗前下降(P<0.05)。ROC曲线分析显示,血清HIF-1α、BNIP3、Beclin-1、S100B水平预测重度TBI的曲线下面积分别为0.782、0.835、0.872、0.880(P<0.05),预测TBI预后不良的曲线下面积分别为0.749、0.775、0.814、0.751(P<0.05)。结论TBI患儿血清HIF-1α、BNIP3及Beclin-1水平显著升高,检测其水平有助于临床判断TBI患儿的病情轻重及预后。

hIL-12及HER2/neu ECD修饰的生孢梭菌工程菌的构建及鉴定

目的构建人IL-12(hIL-12)基因和乳腺癌靶基因HER2/neu胞外区(ECD)分别修饰的生孢梭菌工程菌,探讨其在乳腺癌基因治疗中的作用。方法将hIL-12和HER2/neu ECD的基因分别连接到厌氧梭菌的内源性β-1,4-葡聚糖酶启动子和信号肽序列(eglAp)之后,得到融合基因eglAp-hIL-12和eglAp-HER2/neu。将融合基因插入大肠杆菌-厌氧菌穿梭质粒pIMP1,得到重组质粒pIMP1-ehIL-12和pIMP1-eHER2/neu。将重组质粒首先转化大肠杆菌DH5α,通过酶切和测序鉴定无误后,再用电穿孔法转化生孢梭菌;利用红霉素抗性筛选阳性克隆,采用菌液PCR和质粒提取鉴定阳性克隆,采用ELISA法对生孢梭菌培养上清中目的产物进行分析。结果酶切和测序结果表明重组质粒内的融合基因eglAp-hIL-12和eglAp-HER2/neu序列和读框正确,菌液PCR、质粒提取和ELISA结果显示hIL-12和HER2/neu ECD基因成功修饰生孢梭菌并表达。经过20多代抗生素压力筛选,重组生孢梭菌仍能稳定携带和表达外源基因。结论成功获得hIL-12和HER2/neu ECD基因分别修饰的生孢梭菌工程菌株,为进一步的抗肿瘤研究奠定基础。

pIRES-PML-RARα-hIL-2重组质粒的构建和表达研究

目的构建PML-RARα融合基因与人白介素-2(hIL-2)基因真核双表达质粒。方法利用RT-PCR技术从NB4细胞的RNA中扩增出PML-RARα融合点附近的部分基因片段,通过RT-PCR扩增Jurkat细胞中的hIL-2基因,并分别将两基因片段连接到pIRES质粒的多克隆位点(MCS)A和B中,构建真核双表达质粒。利用酶切和序列分析方法验证所构建质粒的正确性。将构建成功的重组质粒转染A549细胞,通过RT-PCR检测重组质粒在真核细胞中的转录情况,利用点杂交、ELISA检测目的蛋白的表达情况。结果NheⅠ/MluⅠ和SalⅠ/NotⅠ双酶切证明重组质粒中含有相应大小的PML-RARα基因片段及hIL-2基因,序列分析证明重组质粒中插入片段的碱基序列均完全正确。将所构建的pIRES-PML- RARα-hIL-2质粒转染A549细胞后经RT-PCR鉴定表明,插入到重组质粒中的PML- RARα和hIL-2基因能够在真核细胞中进行正常转录。经点杂交和ELISA检测显示重组质粒在真核细胞中可表达hIL-2蛋白。结论成功构建了pIRES-PML-RARα-hIL-2质粒,该重组质粒能够在真核细胞中正常转录并表达PML—RARα和hIL-2蛋白。

融合基因mGM-CSF/hIL-2在肝癌细胞瘤苗特异性免疫治疗中的作用

背景与目的:手术切除是治疗肝癌的主要方法,但对其术后的复发转移却无更好的办法。近年来,免疫学的发展使肝癌的治疗有了更好的治疗方法。本研究旨在通过制备人白细胞介素2(hIL-2)与鼠粒-单核细胞集落刺激因子(mGM-CSF)融合基因修饰的H22肝癌瘤苗,观察其特异性抗肿瘤免疫作用。方法:用含hIL-2与mGM-CSF融合基因的真核表达载体,在体外转染H22细胞,制成疫苗,皮下接种小鼠,同时建立荷瘤小鼠模型。用51Cr释放法测定瘤苗免疫组、空载组、未转染组小鼠脾细胞对亲本H22细胞的杀伤活性。取血检测血清中IL-10、IFN-γ水平,观察小鼠存活期。结果:成功制备了含有hIL-2与mGM-CSF融合基因的H22肝癌瘤苗。免疫小鼠脾细胞体外对H22细胞的杀伤率为38.3%,显著高于对S180细胞的9.1%,以及空载组和未转染组的13.6%和7.5%(P<0.05)。转基因瘤苗免疫组血清IFN-γ为(12.83±0.75)pg/mL,较空载瘤苗组的(7.83±0.65)pg/mL明显升高(P<0.01),血清IL-10[(4.58±0.34)pg/mL]较空载瘤苗组的(8.15±0.28)pg/mL明显降低(P<0.01)。同时,转基因瘤苗免疫组小鼠存活期为(40±6)d,较对照组[空载瘤苗组(30±3)d,未转染组(19±4)d]明显延长。结论:转染hIL-2与mGM-CSF融合基因的同系肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应,延长荷瘤小鼠存活期。

重组免疫毒素hIL-2-Luffin P1对大鼠肾移植存活时间的影响

目的以人IL-2片段与植物毒素Luffin P1重组获得的免疫毒素hIL-2-Luffin P1,观察它对大鼠肾移植排斥反应的抑制作用。方法采用同种异体大鼠肾移植模型,观察hIL-2-Luffin P1对移植物存活的影响,以及移植物的组织病理学观察。结果hIL-2-Luffin P1组大鼠存活时间延长(13.86±2.11)d,与对照组比较相差显著(P<0.01);而Luffin P1处理组与对照组相比无明显差异。同时,病理结果显示对照组移植肾单位结构破坏,淋巴细胞浸润明显,而在同一时间hIL-2-Luffin P1处理组大鼠的移植肾脏病理变化较轻;T淋巴细胞亚群检测显示经hIL-2-Luffin P1处理后,受体外周血T细胞的活化程度降低。结论hIL-2-Luffin P1能够抑制免疫排斥反应,延长移植物存活时间。

HIV-1中国流行株gag与hIL-2在重组痘苗病毒中的共表达及其与核酸疫苗的实验免疫研究

目的：将HIV-1中国流行株B亚型gag基因、gag和hIL-2基因在天坛株痘苗病毒中进行共表达，以期获得重组痘苗病毒，观察细胞因子的佐剂作用，与核酸疫苗混合免疫，评价免疫效果，为新型艾滋病疫苗研制开发打下基础。方法：将HIV-1中国流行株 gag基因、gag和hIL-2基因片段插入到 pJ38载体启动子下游，经同源重组和血凝素阴性空斑筛选重组痘苗病毒，SDS-PAGE、Western blot检测目的蛋白。以重组病毒和核酸疫苗免疫Balb/c小鼠，进行淋巴细胞转化实验、CTL、CD4+、CD8+ T细胞数目以及血清抗体的细胞免疫和体液免疫指标检测。结果：获得了重组痘苗病毒 vJ38gag和 vJ38gag-IL-2。与 vJ38-gag相比，vJ38gag-IL-2，具有更好的免疫原性，重组痘苗病毒免疫3次的效果好于重组病毒免疫2次，以2rVV-DNA混合免疫效果最好。结论：重组痘苗病毒vJ38gag和vJ38gag-IL-2能够表达外源蛋白并诱导机体产生细胞免疫和体液免疫。细胞因子IL-2发挥了免疫佐剂的作用。

PML-RARα-hIL-2 DNA疫苗免疫BALB／c小鼠后胸腺初始T细胞水平变化

目的了解PML-RARα-hIL-2基因疫苗免疫小鼠后胸腺初始(na(?)ve)T细胞变化情况。方法利用实时定量PCR检测小鼠胸腺组织DNA中T细胞受体删除DNA环(TRECs)的水平,从而评价小鼠胸腺中na(?)ve T细胞含量。结果利用PML- RARα-hIL-2 DNA疫苗免疫小鼠后胸腺中所含的TRECs拷贝数明显高于单个基因疫苗PML-RARα或hIL-2组及对照组。结论所构建的PML-RARα-hIL-2双表达质粒可诱导小鼠胸腺产生na(?)ve T细胞数量增加,可能与产生更强的细胞免疫应答有关。

HIV-1 p24 gag 与hIL-2基因在重组痘苗病毒中的共表达研究

目的：研究 Ｈ Ｉ Ｖ１ｐ２４ ｇａｇ 与 ｈ Ｉ Ｌ２基因在重组痘苗病毒中的共表达。方法：以痘苗病毒复制非必需区血凝素（ Ｈ Ａ）基因为侧翼，将编码人白细胞介素２（ｈ Ｉ Ｌ２）的基因片段克隆到真核表达质粒ｐ１６ Ｑ Ｆ２４ Ｉ Ｌ２，经同源重组和血凝素阴性空斑筛选，获得了重组痘苗病毒ｖ１６ Ｑ Ｆ２４ Ｉ Ｌ２。结果：经间接免疫荧光试验、 Ｄｏｔ Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｗ ｅｓｔｅｒｎ ｂｏｌｔ 等检测证明，重组病毒能同时表达ｐ２４ ｇａｇ 蛋白和 Ｉ Ｌ２蛋白，表达产物分子量分别为２４ ０００、１６ ０００。结论：这种重组痘苗病毒人表达外源蛋白的成功，为获得更强的免疫效果，研制 Ｈ Ｉ Ｖ 重组病毒活疫苗提供一种安全的新途径。

PML-RARα-hIL-2 DNA疫苗诱导BALB／c小鼠的细胞和体液免疫应答

目的分析PML-RARα融合基因疫苗诱导小鼠特异体液和细胞免疫应答情况。方法用成功构建的重组质粒(pIRES-PML-RARα-hIL-2)免疫小鼠后,分别利用ELISA法检测小鼠血清中INF-γ生成情况;LDH释放法检测小鼠脾细胞特异性CTL细胞针对APL细胞株NB4的杀伤率;用间接免疫荧光法检测血清中抗PML-RARα抗体。结果免疫后第7周时,小鼠血清中INF-γ含量、抗PML-RARα抗体及脾脏CTL细胞针对NB4的杀伤率均高于对照组。结论所构建的pIRES-PML-RARα-hIL-2重组质粒可诱导小鼠产生了特异性体液和细胞免疫应答。

腺病毒介导hIL-2基因转染膀胱上皮的体内研究

目的:探讨腺病毒介导人IL-2基因转染鼠膀胱移行上皮细胞及其表达的可行性,为膀胱癌的免疫治疗提供实验依据。方法:通过膀胱灌注的方法将重组腺病毒AdhIL-2灌入大鼠膀胱内,分不同时间段获取大鼠膀胱黏膜组织,利用RT-PCR的方法检测hIL-2基因在膀胱移行上皮细胞中的表达。结果:腺病毒介导hIL-2在膀胱移行上皮细胞中得到有效表达,基因表达持续时间达到7天。结论:腺病毒是一种有效介导基因的工具,hIL-2在膀胱上皮细胞中的持续表达为膀胱癌免疫治疗提供新的方法。

重组腺病毒载体pAdBM5-GFP-hIL-2的构建及病毒滴度测定

目的:构建重组腺病毒载体pAdBM5-GFP-hIL-2,并测定病毒滴度。方法:从Jurkat细胞中提取mRNA,设计特异性引物,通过RT-PCR法扩增hIL-2基因全编码区序列。将测序正确的片段用BglII和PmeI双酶切定向插入到pAdBM5-GFP腺病毒载体中。通过磷酸钙共沉淀法将线性化重组质粒和腺病毒骨架共转染HEK293A细胞,扩增纯化重组腺病毒,TCID50法测定重组腺病毒滴度。结果:成功构建出表达hIL-2基因的重组腺病毒载体(pAdBM5-GFP-hIL-2),获得了高滴度表达hIL-2基因的重组腺病毒。结论:重组腺病毒表达载体(pAdBM5-GFP-hIL-2)的成功构建及重组腺病毒的获得有利于进一步开展肝癌的转基因治疗研究。