逆转录病毒载体介导大鼠乳腺癌细胞共表达MIP1α和B71

目的：建立共表达小鼠MIP-1α和B7-1基因的大鼠乳腺癌细胞株，并进行体外活性检测。方法：应用含不同选择标记的重组逆转录病毒载体，经1mg／ml G418和2rLg／ml puromycin双药物筛选后获得MIP-1α＋B7-1基因共表达的大鼠乳腺癌细胞株。RT-PCR、免疫组化检测mMIP-1α的表达，RT—PCR、流式细胞术检测mB7-1的表达；将共表达MIP-1α和B7-1的肿瘤细胞与大鼠脾淋巴细胞混合培养后，MTT法检测淋巴细胞的增殖指数、琼脂糖打孔法检测共表达MIP-1α和B7-1肿瘤细胞对单个核细胞的趋化活性。结果：经脂质体转染包装细胞产生的重组逆转录病毒上清病毒滴度达4．6×10^7CFU／L。细胞生长曲线显示，重组逆转录病毒感染对乳腺癌细胞增殖无明显影响。重组逆转录病毒感染的大鼠乳腺癌细胞株SHZ-88／mMIP-1α＋mB7—1有B7-1和MIP-1αmRNA及蛋白的表达。淋巴细胞增殖指数PI值SHZ-88／PLXSN组为（0．76±0．25），SHZ-88／mMIP-1α＋mB7-1组为（1．95±-0．31），后者体外刺激淋巴细胞增殖能力增强（P〈0．01），SHZ-88／pBabe puro组的趋化指数为（0．99±-0．19），mMIP-1α＋mB7—1组的为（3．88±0．33），后者显著增强了趋化活性。结论：通过逆转录病毒载体介导建立MIP-1α和B7-1基因共表达的大鼠乳腺癌细胞株具有招引单个核细胞，并将其激活的体外生物学活性。     

肝癌患者A-LAK细胞与苯乙酸协同抗瘤的观察

目的：构建重组人FN多肽的真核表达载体,研究体内表达对免疫细胞的趋化作用及抑制肿瘤的生长的作用。方法：采用重组DNA技术构建表达质粒;体内外进行基因转染;用Western blot方法鉴定表达产物;肌肉组织切片与染色观察体内基因转染后的趋化作用;小鼠实体瘤模型研究基因转染抑制肿瘤生长的作用。结果：将人FN cDNA5'端非编码区及信号肽编码区、CH50多 编码区cDNA、人FN cDNA的3'端非编码区重组连接并插入pcDNA3.1质粒,构建出pCH510。以pCH510转染小鼠NIH3T3细胞,可以表在产生CH50多肽。肌肉内注射转染pCH510可对免疫细胞产生趋化作用,抑制实体肿瘤的生长。结论：质粒pCH510可在细胞中及小鼠体内表达;体内表达可对免疫细胞产生趋化作用,并可抑制实体瘤生长。     

TRAIL真核表达治疗肝细胞癌作用的研究

目的：构建鼠源TRAIL（mTRAIL）真核表达质粒。研究其体内、外表达对肝癌细胞的诱导凋亡作用，抑制肝癌生长作用及与重组人FN多肽真核表达质粒pCH510协同抑制肝癌生长作用。方法：采用RTPCR及重组DNA技术构建mTRAIL真核表达质粒；体内、外进行基因转染；用流式细胞仪检测肝癌细胞凋亡率，用TdTmediated dUTP nick end labeling (TUNEL)法及免疫组织化学技术检测肝癌细胞凋亡；小鼠实体瘤块模型研究基因转染抑制肝癌生长作用。结果：用RTPCR方法自小鼠脾细胞RNA扩增出TRAIL基因的全长cDNA，并克隆至真核表达载体pcDNA3.1中获重组质粒pX1；以pX1转染BHK细胞株后攻击肝癌细胞株H22细胞，可检测到肝癌细胞凋亡；肌肉内注射转染质粒DNA pX1，通过诱导肿瘤细胞凋亡抑制肝癌生长；pX1与FN多肽真核表达质粒pCH510有协同抑制肝癌生长作用。结论：质粒pX1可在细胞及小鼠体内表达；体内、外表达可诱导肝癌细胞凋亡并可通过该机制抑制肝癌生长；与FN多肽真核表达质粒pCH510有协同抑制肝癌生长作用。     

复制缺陷型重组腺病毒介导mlFN-β基因治疗小鼠黑色素瘤的研究

目的：探讨人类复制缺陷型重组腺病毒载体介导mIFN-β基因治疗小鼠黑色素瘤的可行性和效果。方法：利用人类复制缺陷型重组腺病毒将目的基因mIFN-β经体外、体内两种途径导入小鼠黑色素瘤细胞（B16细胞）中，观察体外转mlFN-β基因的B16细胞的体内致瘤性和瘤苗作用，及通过腺病毒载体介导的mIFN-β对体内局部肿瘤治疗和抗肿瘤转移的作用。结果：1）携带目的基因的重组腺病毒感染B16细胞能获得较高的转移效率和目的基因的有效表达；2）将mIFN-β基因导入B16细胞对B16细胞的体外增殖能力无明显影响，但能显著提高细胞表面MHC-I类抗原的表达；3）将转mIFN-β基因的B16细胞接种小鼠，其体内致瘤性明显降低．且对对侧野生型B16细胞的致瘤性有抑制作用；4）瘤体内注射和经尾静脉注射途径给予AdCMV mIFN-β．对局部肿瘤和转移瘤有治疗作用。结论：利用人类复制缺陷型腺病毒载体介导mIFN-β基因治疗小鼠黑色素瘤是可行的，并具有较好疗效，提示用腺病毒载体携带有效的目的基因来开发瘤苗和治疗肿瘤具有良好的临床应用前景。     

HPV16Z-Hsp65-E6／E7无佐剂重组蛋白疫苗的研究

目的：研究治疗性HPV16Z-Hsp65-E6／E7无佐剂重组蛋白疫苗的抗肿瘤活性。方法：通过淋巴细胞增殖实验和细胞毒性杀伤实验研究该疫苗激发的细胞免疫反应及反应强度；观察该疫苗对小鼠TC-1肿瘤细胞移植瘤的体内治疗作用和对小鼠生存期的影响。结果：重组蛋白疫苗免疫小鼠后，小鼠脾淋巴细胞与该疫苗体外混合培养增殖明显，并可特异性地在体外杀伤TC-1细胞；体内抑瘤试验显示该疫苗对HPV16病毒转化的TC-1细胞小鼠移植瘤的生长有显著的抑制作用。结论：该疫苗能激发特异性细胞免疫反应；显著抑制HPV16转化的TC-1肿瘤细胞生长。     

B7基因修饰的肿瘤细胞疫苗体内外调控细胞因子水平的研究

我们率先在国内外报道了有关B7基因修饰肝癌细胞对其免疫原性和致瘤性的影响及B7基因修饰的肿瘤细胞疫苗(tumor cell vaccine,TCV)的体内、外抗肿瘤作用.我们的研究发现,在缺乏B7分子的小鼠肝癌细胞Hepal-6与细胞株中导入小鼠B7-1,B7-2基因,能使该肝癌细胞株的免疫原性大大增强,致瘤性完全消失.用丝裂霉素C(MMC)处理转染了B7-1,B7-2基因的小鼠肝癌细胞Hepal-6细胞株,制备成肿瘤细胞疫苗,其抗肿瘤作用明显增强,表现为对论动物的免疫模型起完全的免疫保护作用、对早期模型起部分治疗作用和     

应用细胞因子缓释微球的肿瘤疫苗治疗肝癌的研究

目的 探讨采用细胞因子缓释微球的肿瘤疫苗预防和治疗肝癌的疗效及抗癌机制。方法 我们研制开发了一种肿瘤疫苗,其组成是固定的肿瘤细胞或组织碎片、细胞因子缓释微球和免疫辅助药。采用多聚甲醛固定的小鼠Hepal-6细胞或肿瘤碎片、微球包装的GM—CSF和／IL—2和合成TiterMax Gold等不同成份的瘤苗皮内接种C57BL／6J小鼠,随后肝内接种活体Hepal-6细胞。结果 对照组15只小鼠全部发展成肝肿瘤;含有固定Hepal-6细胞和IL-2及GM—CSF微球的肿瘤疫苗,80％小鼠获得保护。再加入免疫辅助剂TiterMax Gold的肿瘤疫苗,则87％小鼠获得保护。将Hepal-6细胞接种于左躯干皮下。肿瘤长至直径5mm时,皮内接种肿瘤疫苗2次。结果显示,对照组肿瘤继续生长。疫苗组在第2次接种后7—10天,10只小鼠中9只肿瘤生长受到抑制,随后明显缩小。60％小鼠的肿瘤完全消散。细胞毒性实验结果显示,未接种疫苗的小鼠脾细胞不能杀灭Hepal-6细胞和其他肿瘤细胞;而接种疫苗的小鼠脾细胞对Hepal-6细胞杀瘤活性达41％,但对B16—Fl,Lewis肺癌细胞(LLC),肾癌细胞(Renca),膀胱癌细胞(MBT-2)则无效。疫苗的Ⅰ期临床实验结果显示肝癌疫苗能有效地预防肝癌术后复发,诱导DTH反应。结论 肝癌疫苗能有效预防和治疗原发性肝癌,其抗瘤机制是诱导内源性抗原特异性CTL反应,其杀瘤特性是由典型的：MHC—Ⅰ限制的CD8^ T细胞所介导的。     

表达白细胞介素-12质粒DNA抗小鼠肝癌皮下移植瘤的作用

目的 探讨瘤内注射mIL-12质粒DNA抗小鼠肝癌皮下移植瘤的作用。方法：构建真核表达质粒载体pDC511mIL-12，ELISA方法检测质粒载体在真核细胞中的表达，淋巴母细胞增殖法检测mIL-12的生物学活性；分别于小鼠肝癌H22皮下移植瘤内直接注射质粒DNA，观察各组小鼠存活时间、肿瘤体积变化及各组小鼠脾脏细胞毒T淋巴细胞(CTL)的活性：注射质粒DNA后1月进行瘤体组织病理学观察：结果：mIL-12基因治疗组与空载体对照组相比，肿瘤生长显著受抑制(F=4．10，P=0．03)，小鼠存活期显著延长(X^2=4．48，P=0．03)．并且小鼠脾细胞CTL杀伤活性增强。质粒DNA瘤内注射1月后，pDC511mIL-12组肿瘤病灶炎性细胞浸润明显，病灶内肿瘤细胞广泛坏死。结论：瘤内注射mIL-12表达质粒DNA可抑制小鼠肝癌皮下移植瘤生长，能提高机体抗肿瘤免疫应答。     

负载人AFP肽段的树突细胞在体内外对小鼠肝癌的抑制作用

背景与目的:甲胎蛋白(alpha-fetoprotein,AFP)是原发性肝细胞癌(hepatocellular carcinoma,HCC)免疫治疗的一个良好的靶分子,如何克服对自身抗原的免疫耐受状态是诱导有效抗肿瘤免疫反应的关键.本研究探讨异种同源蛋白疫苗负载人AFP肽段的树突细胞(human AFP-derived peptide-pulsed dendritic cells,hAFP-DCs)对小鼠肝癌的体外杀伤作用和体内抑瘤效应.方法:传统方法制备骨髓来源的DCs.MTT法检测hAFP-DCs诱导的CTL对小鼠肝癌细胞Hepal-6的体外杀伤活性.建立Hepal-6细胞C57BL/6小鼠移植瘤模型,分别瘤内注射hAFP-DCs、DCs和PBS(每周两次),观察小鼠肿瘤体积和荷瘤存活时间.结果:成功制备小鼠骨髓来源的DCs.体外杀伤实验显示,hAFP-DCs刺激组和单纯DCs刺激组CTL对Hepal-6细胞的杀伤作用强于PBS组,但组间差异无统计学意义(P>0.05).体内实验表明.每个C57BL/6小鼠接种7×106个Hepal-6细胞31 d后,hAFP-DCs、DCs和PBS组小鼠平均移植瘤体积分别为(195.04±155.22)mm3、(360.65±209.02)mm3和(756.19±503.24)mm3,组间比较差异有显著性(P<0.001).在40 d的观察期内,小鼠的累积存活率分别为100%、90%和50%(P=0.008).结论:负载人AFP抗原肽的DEs疫苗在体外和体内均能有效抑制小鼠肝癌的生长.     

外源性野生型p53基因抑制裸小鼠体内人白血病细胞的生长

目的探讨野生型ｐ５３基因对体内生长的白血病细胞的抑制作用。方法采用直接注射法向裸小鼠体内ＨＬ６０－ｎ和Ｋ５６２－ｎ细胞移植瘤注射编码人野生型ｐ５３的重组逆转录病毒，使其在这两种细胞中表达，并导致其编程性细胞死亡。结果裸小鼠在接种ＨＬ６０－ｎ或Ｋ５６２－ｎ细胞后２４ｈ，于接种部位连续７天注射编码野生型ｐ５３的逆转录病毒，移植瘤发生的潜伏期较对照组明显延长。结论直接注射转移野生型ｐ５３基因能有效地抑制裸小鼠体内人白血病细胞的生长。     

INVESTIGATION OF THE THERAPEUTIC EFFECT OF EXPRESSION OF TRAIL IN VIVO ON MOUSE HEPATOCELLULAR CARCINOMA

Objective: To construct an eukaryotic expressing plasmid of mouse TRAIL (mTRAIL), and investigate its ability to induce the apoptosis of hepatocellular carcinoma cells in vitro and in vivo, its inhibitory effect on the growth of hepatocellular carcinoma, and its synergism with pCH510, an eukaryotic expressing plasmid of recombinant human FN polypeptide.Methods: The eukaryotic expressing plasmid of mTRAIL was constructed by RT-PCR and DNA recombination techniques. Gene transfection was performed in vitro and in vivo. The apoptosis rate of hepatocellular carcinoma cells was measured by Flow Cytometry. The apoptosis of hepatocellular carcinoma cells was measured by Flow Cytometry.The apoptosis of hepatocellular carcinoma cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) and histochemistry techniques. The inhibitory effect of gene transfection on solid tumor was observed in mice. Results: The cDNA of mTRAIL was amplified by RT-PCR from the RNA of mouse spleen cells, and cloned into the eukaryotic expressing vector pcDNA3.1. The recombinant plasmid was designated as pX1. The BHK cells transfected with plasmid pX1 could attack H22 hepatocellular carcinoma cells and induce the apoptosis of them. The transfection of plasmid pX1 through injection into mouse muscles could inhibit the growth of hepatocellular carcinoma by inducing the apoptosis of tumor cells. Plasmid pX1 and pCH510 had a synergistic inhibitory effect on the hepatocellular carcinoma growth. Conclusion: Plamid pX1 could be expressed in cells and in vivo in mouse. The expression of pX1 in vivo and in vitro could induce the apoptosis of hepatocellular carcinoma ceils and inhibit the growth of hepatocellular carcinoma. Plasmid pX1 and pCH510 had a synergistic inhibitory effect on the hepatocellular carcinoma growth.     

Transduction of antisense cyclin D1 using two-step gene transfer inhibits the growth of rat hepatoma cells.

Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.     

重组人GM—CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定…

建立逆转录病毒介导的ＧＭ－ＣＳＦ基因转移系统,为ＧＭ－ＣＳＦ基因悠我肿瘤细胞疫苗研究奠定实验基础。应用基因重组技术将ＧＭ－ＣＳＦｃＤＮＡ克隆于逆转录病毒载体ｐＤＯＲｎｅｏ,获得重组载体ｐＤＯＲＧＭ。经脂质体介针其转染于病毒包装细胞系ＰＡ３１７细胞,经ＮＩＨ３Ｔ３细胞检测病毒滴度,ＮＩＨ３Ｔ３放大实验检测辅助病毒,并用同熵度病毒感染人乳腺癌细胞系ＭＣＦ－７。     

肝癌细胞绿色荧光蛋白基因的转染与克隆化培养

目的：研究建立稳定高表达绿色荧光蛋白（ＧＦＰ）并能连续传代培养的肝癌细胞株。方法：将携带ＣＦＰｃＤＮＡ的重组逆转录病毒载体转染肝癌细胞株７７２１,经过Ｇ４１８筛选和克隆化培养,用荧光显微镜检测癌细胞荧光表达。结果：转染ＧＦＰ ｃＤＮＡ的癌细胞５ｄ后大部分死亡,荧光显微镜下有散在或小团状的点状荧光。继续用Ｇ４１８筛选及克隆化培养６５ｄ后第２８培养孔的癌细胞几乎均见荧光表达并可连续稳定传至１５例以上的     

小鼠AFPcDNA真核表达载体的构建、表达及体外抗肝癌免疫活性的检测

背景与目的:探索肝癌细胞的突变基因产物——具有潜在抗原性的异常蛋白质而无法形成有效免疫原的机理,寻找肝癌的特异性抗原,并在此基础上研制出治疗肝癌的DNA疫苗将对肝癌的免疫学治疗产生积极的影响。本实验构建BALB/c小鼠具有分泌性信号肽的AFP1cDNA和去掉信号肽的AFP2cDNA真核表达载体,分别在小鼠树突细胞(dendriticcells,DCs)中表达,并观察其在体外抗肝癌免疫中的作用。方法:采用RT-PCR方法自小鼠肝癌细胞株H22中扩增出具有分泌性信号肽的AFP1cDNA和去掉分泌性信号肽的AFP2cDNA,将该基因定向插入真核表达载体PEGF-N3中;同时培养经rmGM-CSF、rmIL-4诱导的小鼠骨髓细胞,体外获取大量DCs,脂质体转染PEGF-N3/AFP1、PEGF-N3/AFP2至DCs进行表达、鉴定。将DCs疫苗与同源小鼠脾淋巴细胞混合培养,ELISA方法测定脾细胞γ-干扰素(IFN-γ)分泌活性,51Cr释放法测定脾淋巴细胞的特异性杀伤活性。结果:从H22中克隆到AFP1cDNA和AFP2cDNA,经测序完全正确,所构建的真核表达质粒PEGF-N3/AFP1和PEGF-N3/AFP2在小鼠DCs中获得稳定高效表达,其中PEGF-N3/AFP2明显刺激T细胞增殖,刺激指数(SI)为5.12±1.46,明显高于空质粒组(1.42±0.73)。AFP2/DC疫苗对H22细胞的特异性杀伤活性[(88.15±16.47)%]明显高于空质粒组[(12.72±5.45)%]。结论:成功地构建了真核表达载体PEGF-N3/AFP1和PEGF-N3/AFP2,编码去掉分泌性信号肽的PEGF-N3/AFP2转染的DC,能够诱导较强的特异性抗肝癌免疫效应。其机制可能为诱导肝癌细胞凋亡。     

Improvement of combination chemotherapy tolerance by introduction of polycistronic retroviral vector drug resistance genes MGMT and MDR1 into human umbilical cord blood CD34+ cells.

We obtained a full-length cDNA fragment encoding human O(6)-methylguanine-DNA-methyltransferase (MGMT) from the liver tissue of a patient with cholelithiasis by RT-PCR and confirmed by DNA sequencing. The polycistronic retrovirus vector G1Na-MGMT-Neo(r)-IRES-MDR1 was constructed and verified by restriction endonuclease analysis and DNA sequencing. The vector was transfected into packaging cells GP+E86 and PA317 by the LipofectAMINE method. Cord blood CD34+ cells were transfected with the supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hematopoietic growth factors. PCR, RT-PCR, Southern Blot, Western Blot, FACS and MTT analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 5.8-6.3-fold stronger resistance to P-glycoprotein effluxed drugs and 5-fold to BCNU than untransduced cells. The polycistronic retrovirus vector mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.     

Retroviral Introduction of the p16 Gene into Murine Cell Lines to Elicit Marked Antiproliferative Effects

The pl6 gene is a candidate tumor suppressor, because mutation of the gene has been reported in many transformed cell lines and some primary tumor tissues. We have examined this possibility in murine cell lines (NIH3T3 and RSV-M) which lack pl6 gene expression. Full-length human p16 cDNA was obtained from a HeLa cell line using polymerase chain reaction amplification. We constructed two separate retrovirus vectors carrying this pld cDNA. First, we transduced the p16 cDNA into the murine cell lines using a retrovirus vector harboring the neomycin-resistance gene. The p16 genetransduced cells formed no colonies after selection with G418, in contrast to the vector-transduced cells. Next, we used another retrovirus vector that expresses both the p16 cDNA and the Lac Z gene, which enabled us to distinguish affected cells from unaffected ones. Proliferation of the p16 gene-transduced cells was markedly inhibited and morphological change in the cells was also observed. Thus, we concluded that the p16 gene has an antiproliferative effect on the cell cycle and that the loss of its function may play a major role in dysregulated proliferation of the cells.     

基因转染法建立bcl-2稳定表达的肝癌细胞株

目的与方法：为了建立稳定表达 bcl-2蛋白的肝细胞癌（简称肝癌）细胞株,用脂质体介导的基因转染法将含有人 bcl-2,CDNA的逆转录病毒表达载体pDOR-SB质粒导入bcl-2蛋白阴性的肝癌细胞系HCC－9204细胞中,经G－418筛选后获得转 bcl-2基因的细胞克隆。细胞扩大培养后用免疫细胞化学法检测bcl-2表达情况,有限稀释法连续克隆化直至获得100％稳定表达bcl-2蛋白的单克隆细胞株,并进行流式细胞仪检测,结果：转染了pDOR-SB的HCC－9204细胞大部分为bcl-2蛋白表阳性,而转染了pDOR空载体或未转染的HCC－9204细胞均为bcl-2蛋白阴性,经过连续3次克隆化后,流式细胞仪检测表明所获得的单克隆细胞 100％表达bcl-2蛋白,结论：用基因转染法成功地建立了稳定表达bcl-2蛋白的肝癌细胞株。