B7-1转基因骨肉瘤疫苗的制备及鉴定

目的构建含B7—1基因与绿色荧光蛋白（GFP）基因的融合表达载体,转染LM8骨肉瘤细胞制备骨肉瘤疫苗。方法用RT—PCR法扩增B7—1基因片断,应用含GFP基因的真核表达质粒构建融合表达载体,酶切、测序鉴定构建质粒;采用脂质体介导方法将其转染到LM8细胞,并用荧光显微镜及LCM结合蛋白免疫印记检测其在肿瘤疫苗细胞中的表达。结果经PCR及酶切鉴定,证实成功构建了含B7—1基因的真核表达重组体pEGFP—C1／B7,重组子测序结果与国际基因文库中mB7—1序列相符。用荧光显微镜观察到疫苗细胞中有GFP表达,RT—PCR检测到疫苗细胞中B7—1基因的表达．westernblot发现疫苗细胞中有B7蛋白的表达。结论B7—1重组真核绿色荧光表达载体pEGFP—C1／B7已成功构建,转染,LM8细胞成功制备了骨肉瘤细胞疫苗。     

人B7-2和增强型绿色荧光蛋白融合基因表达载体的构建

目的构建含人B7-2与增强型绿色荧光蛋白融合基因表达载体pEGFP-N3-B7-2.方法应用逆转录聚合酶链反应(RT-PCR)方法从重症肌无力患者胸腺组织中扩增到人B7-2cDNA基因片段,经回收、纯化、酶切后,依次连接到质粒pGEM-Teasy和pEGFP-N3上.结果通过酶切和序列分析证实插入片段序列正确.结论应用基因工程技术构建成功pEGFP-N3-B7-2融合基因表达载体,为增强型绿色荧光蛋白作为人B7-2生物标记分子,转染肿瘤细胞及树突状细胞制备疫苗奠定了基础.     

mB7-1修饰的B16细胞体外诱导免疫效应

目的 探讨mB7-1基因修饰的B16细胞的体外抗瘤效应.方法 提取小鼠脾细胞mRNA,PCR法获得mB7-1片段,与pcDNA3连接,并进行酶切鉴定及测序.重组质粒经脂质体介导转染鼠B16细胞,检测基因表达,MTT法检测CTL杀伤作用.结果 完成mB7-1-pcDNA3重组载体连接及鉴定,基因测序与GenBank一致,并转染至B16细胞,检测到基因表达.经mB7-1修饰B16细胞可诱导淋巴细胞产生明显抑瘤效应.结论 应用本文方法 可获得mB7-1修饰B16细胞系,为研究其抗瘸效应莫定了基础.     

HSV-TK逆转录病毒包装细胞系建立及病毒滴度测定

目的 建立HSV-TK逆转录病毒包装细胞系并获得高产病毒的细胞株.方法 将TK基因与逆转录表达载体PLEGFP-N1连接后转染到包装细胞PA317中,经G418及荧光蛋白双重筛选得到高产病毒的细胞株.结果 经酶切鉴定成功构建PIEGFP-N1-TK重组载体,含HSV-TK基因重组逆转录病毒包装细胞PA317成功建立,经病毒滴度测定获得高产病毒的PA317/TK细胞株.结论 制备的重组病毒具有感染靶细胞的活性,为进一步应用HSV-TK基因进行肺癌的自杀基因治疗研究奠定基础.     

smad7和uPA双基因共表达重组腺病毒载体的构建和鉴定

目的通过引入内部核糖体进入位点序列,构建并鉴定smad7和uPA基因共表达的腺病毒载体.方法PCR扩增uPA和smad7全长cDNA的片段,先后亚克隆入pIRES质粒;更换酶切位点后,将smad7-IRES-uPA片段克隆至穿梭质粒pAdTrack-CMV,再与pAdEasy-1质粒在RJ5183菌中同源重组产生腺病毒载体质粒.酶切鉴定后在293细胞中包装成重组腺病毒Adsmad7/uPA.RT-PCR检测Adsmad7/uPA在L02肝细胞中的表达.结果该重组腺病毒质粒经测序、酶切鉴定,均与预期结果一致;转染AD-293细胞后,2天可观察到GFP明显表达;RT-PCR检测到转染后的L02细胞中smad7和uPA表达增强.结论成功构建了smad7和uPA双基因共表达重组腺病毒载体,为研究mad7和uPA双基因共表达对大鼠肝纤维化的预防、治疗作用奠定基础.     

人Tum-5基因逆转录病毒载体及包装细胞株的构建

目的 构建携带Tum-5基因的逆转录病毒载体并对其进行包装,获得稳定的产毒细胞系.方法 采用RT-PCR法从胎肾组织中扩增Tum-5基因片断,并将其定向克隆到逆转录病毒载体pLXSN中进行PCR、双酶切和测序鉴定.利用电穿孔方法 将获得的重组质粒转染PA317细胞,经G418 筛选抗性克隆,收集病毒上清后感染NIH3T3 细胞测定病毒滴度.结果 PCR、酶切证实Tum-5基因克隆至逆转录病毒载体pLXSN,Tum-5基因测序结果 和原始序列相同.重组逆转录病毒载体转染PA317 包装细胞,RT-PCR证实转染后的PA317细胞上清液中存在携带人Tum-5基因的病毒RNA,病毒滴度为2.05×104cfu/ml.结论 成功的构建了携带人Tum-5基因的逆转录病毒载体,获得了稳定的产毒细胞系.     

人BMP2-IRES-HIF1αmu腺病毒表达载体的构建及其在HEK293细胞中的表达

目的构建人BMP2-IRES-HIF1αmu腺病毒表达载体,并转染HEK293细胞,为下一步转染骨髓基质细胞和体内实验打下基础。方法 PCR扩增HIF1αmu片段,用BstXⅠ和XbaⅠ双酶切回收目的片段。pIRES2-EGFP用BstXⅠ和XbaⅠ进行双酶切后回收大片段。将上述回收的目的基因与载体片段连接,然后转化感受态大肠杆菌DH5α扩增;PCR扩增BMP2片段,用NheⅠ和BamHⅠ双酶切后回收目的片段。把目的基因与载体片段连接,转化感受态大肠杆菌DH5α扩增重组腺病毒表达载体,通过酶切分析、PCR和测序进行鉴定。将构建好的质粒转染HEK293细胞,检测病毒液滴度。结果构建了人BMP2-IRES-HIF1αmu腺病毒表达载体,转染HEK293细胞见绿色荧光表达。结论成功构建了人BMP2-IRES-HIF1αmu腺病毒表达载体,酶切分析及DNA测序证实质粒构建正确,质粒成功转染HEK293细胞,并见绿色荧光蛋白表达。     

染色体脆性位点抑癌基因FHIT真核表达质粒的构建

目的构建人染色体脆性位点抑癌基因FHIT真核表达质粒。方法以通过全基因合成的FHIT cDNA为模版,用PCR技术扩增,扩增片段用BamHI/Xho I双酶切后克隆到真核表达载体pcDNA3.1中,用DNA测序法鉴定重组质粒。结果质粒DNA测序显示与文献报道的人FHIT cDNA序列一致。结论成功构建出含人FHIT基因的真核表达质粒。     

CCR5反义RNA重组表达载体的构建及鉴定

目的构建趋化因子受体CCR5反义RNA真核表达载体以用于抗HIV-1的研究.方法用RT-PCR从健康人外周血单个核细胞(PBMCs)中获得趋化因子受体CCR5翻译起始区的基因片段,用基因重组技术将此目的基因以正、反两个方向定向插入到逆转录病毒载体pLXSN.用PCR、内切酶分析及序列测定鉴定.重组载体用脂质体转染剂(lipofectAMINE)转染PA317包装细胞,建立稳定表达重组假病毒颗粒的包装细胞系.结果从正常人PBMCs中获得的目的基因成功地构建了CCR5反义RNA重组表达载体,重组载体已在PA317细胞中得到整合.结论构建的CCR5正、反义RNA表达载体可有效地感染PA317细胞并在基因组中整合,为进一步研究CCR5反义RNA的抗HIV-1的作用奠定了基础.     

Expression of costimulatory molecules, B7-1 and B7-2 on human gastric carcinoma

Costimulation of T cells via B7-1 and B7-2 molecules on a tumor has been shown to be important for eliciting cell-mediated antitumor immunity. We studied the surface expression of B7-1 and B7-2 in 24 cases of gastric carcinoma from the primary locus, 20 cases of metastatic carcinoma from malignant ascites, 20 cases of benign gastric mucosa and 7 gastric carcinoma cell lines by two-color flow cytometry with mAb CD80 and CD86. The B7-1 and B7-2 molecules were expressed by 6 cell lines, and 1 cell line showed the predominant expression of B7-2 but not B7-1. Almost all patients with primary gastric carcinoma and benign gastric mucosa showed high levels of expression of the B7-1 and B7-2, revealing approximately 40%–60% positive cells. However, the percentage of B7-1-positive cells of poorly differentiated primary carcinomas was significantly lower than that of well-differentiated carcinoma and normal mucosa (P<0.01). Furthermore, all of the metastatic carcinoma cells revealed consistently very low or undetectable levels of expression of the B7-1 molecule, only 8% (mean) of cells being positive, despite showing higher levels of B7-2 expression. Thus, it seems likely that decreased or deleted expression of B7-1 correlates with the grade of tumor differentiation, tumor progression and metastasis. These results suggest that the B7-1 molecule on the gastric carcinoma bearing CD80⁺CD86⁺ is abrogated during tumor invasion and/or metastasis, and the tumor finally acquires the CD80⁻CD86⁺ phenotype. Consequently, inadequate B7-1 costimulation may contribute to the escape of tumors from destruction by the host's immune system. Received: 26 December 1997 / Accepted: 2 March 1998     

B7-2对肝细胞癌特异性免疫的影响

T细胞的激活需要两种信号刺激，第二信号为非抗原特异性，主要由协同刺激分子B7-2家族介导产生。我们将B7-2真核表达载体转染高度表达组织相容性复合物I(MHC-I)分子而缺乏MHC-Ⅱ分子的肝癌细胞株HMC7721，观察B7-2对CD8^ T细胞分化和肿瘤特异性免疫的影响。     

α-干扰素治疗慢性乙型肝炎患者肝组织B7-1表达的变化

目的了解α-干扰素(-αIFN)治疗慢性乙型肝炎(chronic hepatitis B,CHB)患者前后肝组织B7-1的表达特点。方法采用免疫组织化学方法检测18例正规使用-αIFN的CHB患者治疗前、后肝活检组织共刺激分子(B7-1)的表达及其与-αIFN疗效的关系。结果18例CHB患者肝组织B7-1阳性表达颗粒的平均吸光度值治疗后较治疗前显著增加(分别为24.98±4.23和11.77±2.35,t=11.58,P<0.01)。B7-1表达水平与-αIFN近期疗效呈显著正相关(r=0.68,P<0.01)。结论-αIFN可促进肝细胞B7-1表达,B7-1表达水平可能是预测-αIFN近期疗效的重要参数之一。     

B7-1在乙型肝炎肝组织的表达及其与病变的关系

目的阐明Ｂ７－１在乙型肝炎肝组织中的表达及其与病变发生、发展的关系。方法用免疫组织化学方法检测连续切片的乙型肝炎肝组织Ｂ７－１及表面抗原表达。结果正常对照２例均为阴性；１７例慢性迁延性肝炎（ＣＰＨ）中，胞浆Ｂ７－１阳性的有１６例，轻度阳性８例，中度５例，重度３例；１４例慢性活动性肝炎（ＣＡＨ）全部阳性，其中轻度阳性２例，中度３例，重度９例，经等级秩和检验，两者差异显著（Ｐ＜００１）。急性黄疸性肝炎、亚急性重型肝炎轻度阳性。在３４例乙型肝炎肝组织中，３３例ＨＢｓＡｇ阳性，轻度阳性３例，其余为中到重度阳性，且ＣＰＨ与ＣＡＨ两组之间无统计学差异。结论（１）乙型肝炎病毒感染可诱导Ｂ７－１在肝组织表达，且表达强弱与肝炎病变程度正相关；（２）乙型肝炎表面抗原在组织中表达强弱与Ｂ７－１表达无关。（３）Ｂ７－１在肝组织中的表达可能是肝细胞活化积极参与免疫反应的佐证。     

慢性乙型肝炎肝组织B7-1表达与α-干扰素近期疗效的关系

目的了解慢性乙型肝炎(CHB)肝组织B7-1表达与α-干扰素(α-IFN)近期疗效的关系。方法对68例使用α-IFN的CHB患者治疗前行肝组织活检,采用免疫组织化学方法观察其B7-1的表达。结果68例CHB中45例(66.2％)B7-1表达阳性,5例正常人肝组织无表达。α-IFN的总应答率,肝组织B7-1表达阳性组为66.7％(30/45),阴性组为39.1％(9/23),二者差异显著(x2=7.20,P<0.01)。随B7-1阳性表达程度的增强,肝组织炎性活动积分(HAI)及血清丙氨酸转氨酶(ALT)异常值明显增加。结论肝组织B7-1表达水平可能是预测α-IFN近期疗效的重要参数之一。     

由血管紧张素(1-7)重新认识RAS系统

对血管紧张素 (1- 7)的研究使我们对肾素 -血管紧张素系统 (RAS)有了更全面的认识。血管紧张素 (1-7)通过特异性的受体发挥生理作用。血管紧张素Ⅰ和血管紧张素Ⅱ经过特异的肽链内切酶变为血管紧张素 (1 -7) ,再经血管紧张素转换酶作用降解为无活性的血管紧张素 (1- 5 )。血管紧张素 (1- 7)具有抗增殖、扩张血管、抗氧化应激及促进纤溶的作用。     

表达大鼠IL-2、B7-1基因CBRH7919细胞的建立

目的:研究携带大鼠IL-2、B7-1目的基因的CBRH7919细胞(即CBRH7919/IL-2/B7-1)体外表达目的基因的能力,方法:RT-PCR扩增Wistar大鼠IL-2、B7-1目的基因,分别克隆至重组逆转录病毒载体pBaBe-puro及pMSCV-neo,构建逆转录病毒载体pBaBe-puro-IL-2及p...     

血管紧张素-(1-7)与心血管疾病

血管紧张素系统（RAS）作为一种循环激素参与血压调节与盐类代谢,并维持内环境稳态.血管紧张素-（1-7）[Ang-（1-7）]是RAS系统中的一个独立代谢物,且是具有生物活性的七肽.Ang-（1-7）分别由血管紧张素Ⅰ及血管紧张素Ⅱ转化而来.近年来的研究发现其作为血管紧张素Ⅱ的拮抗因子,具有舒张血管、利钠利尿、抑制血管平滑肌增生等多种效应,对心血管基础研究及临床实践有重要意义[1].本文就近年血管紧张素-（1-7）与心血管疾病的研究现状作一简要综述.     

B7 molecule mRNA expression in colorectal carcinoma

AIM: To observe the status of tumor-associated B_7 molecule mRNA expression in human colorectal cancer tissue by in situ hybridization. METHODS: The mRNA expression patterns of cancerassociated B_(7-1),B_7H_1,B_7H_2,ICOS in 22 specimens of human colorectal cancer tissue were monitored by in situ hybridization (ISH) with digoxin-iabeled oligonucieotide probes. RESULTS: B_(7-1)B_7H_1,B_7H_2,ICOS mRNA were detected in both cancer cells and tumor infiltrating lymphocytes (TIL). The mRNA expression level of these molecules in tumor cells was higher than that in TIL (0.76±0.54-1.62±0.82 vs 0.38±0.19-0.65±0.33, P<0.001). There was no relationship between expression level of tested B_7 family molecules and patients'sex, age, differentiation status of cancer and regional lymph node metastasis. CONCLUSION: Th2 cytokine predominant in tumor microenvironment might be related to the expression of B_7H_1,B_7H_2 co-signal molecules in tumor cells and TIL.     

Anti-gastric cancer active immunity induced by FasL／B7-1 gene-modified tumor cells

AIM:To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells,and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally,and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z=2.15-46.10, P<0.01).SGC-7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC-7901 cells (z=2.06-44.30, P<0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%, P<0.05). CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.