肺癌抗原负载的树突细胞体外诱导细胞毒性T淋巴细胞的杀瘤作用

目的探讨不同方式肺癌抗原负载的树突状细胞(DC)在抗肿瘤免疫中的作用。方法取肺癌患者外周静脉血体外诱导和培养DC,分别以肺癌细胞总RNA转染DC(转染组)、肺癌细胞融合DC(融合组)和肺癌细胞冻融抗原负载DC(冻融组),以未负载抗原的DC(未负载组)和T细胞组作为对照,比较各组(每组n=6)DC诱导的细胞毒性T淋巴细胞(CTL)对肺癌细胞A_(549)的杀伤率(%)。结果转染组、融合组、冻融组、未负载组和T细胞组的杀伤率分别为(73．2±5．9)%、(61．6±6．2)%、(55．3±6．9)%、(22．3±6．1)%和(19．8±6．3)%(P＜0．05)；其中转染组、融合组和冻融组高于未负载组和T细胞组(P＜0．05)；转染组和融合组之间差异无统计学意义(P＞0．05),但两者均大于冻融组(P＜0．05)。RNA转染DC所需的肿瘤细胞数仅是冻融及融合方式的1/5和1/6。结论3种肺癌抗原负载DC方式均有诱导效应细胞杀伤靶细胞的增效作用,而以肺癌细胞总RNA转染方式为佳。     

树突状细胞激活的肿瘤浸润性淋巴细胞体外特异性抗小鼠肝癌研究

目的树突状细胞(DC)是目前已知的功能最强的抗原提呈细胞(APC),可以向包括肿瘤浸润性淋巴细胞(TIL)在内的T淋巴细胞提呈抗原,并诱发细胞毒T淋巴细胞(CTL)反应。本文旨在探讨H22细胞和B16细胞全细胞性抗原致敏的树突状细胞激活的肿瘤浸润性淋巴细胞体外抗小鼠肝癌活性。方法从小鼠四肢长骨骨髓中获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤全细胞性抗原致敏DC,然后用DC激活TIL,观察TIL在体外对H22细胞、Hepal-6细胞和B16细胞的杀伤活性。结果经H22细胞全细胞性抗原致敏的DC激活的TIL具有很高的对H22细胞杀伤活性,杀伤率为(71·31±3·11)%,明显高于其对Hepal-6和B16细胞的杀伤活性[杀伤率分别为(50·11±3·03)%,(30·31±2·89)%];也明显高于未经H22细胞全细胞性抗原致敏的DC激活的TIL、DC激活的脾淋巴细胞和未经DC激活的脾淋巴细胞对H22细胞杀伤活性[杀伤率分别为(49·80±3·21)%,(48·76±3·60)%和(19·23±2·71)%]和对Hepal-6细胞杀伤活性[杀伤率分别为(39·4±3·21)%,(38·62±2·87)%和(18·73±2·40)%]以及对B16细胞杀伤活性[杀伤率分别为(26·38±2·51)%,(25·82±2·70)%和(18·34±3·01)%],同时经B16细胞全细胞性抗原致敏的DC激活的TIL(来源于H22瘤体)也可诱导相对较低的对B16细胞的特异性细胞杀伤活性。结论来源于H22瘤体的TIL经H22细胞全细胞性抗原致敏的DC激活后可产生很强的针对H22细胞的特异性杀伤活性,明显高于其他各组,说明DC能诱导TIL产生高效而特异的体外抗小鼠肝癌免疫。     

反义IDO-AFP基因融合修饰树突状细胞抗肝癌免疫作用

目的探讨反义IDO-AFP基因融合修饰树突状细胞(DC)后,DC通过调控色氨酸代谢达到高效、特异性抗肝癌作用。方法采用细胞培养方法,从小鼠骨髓中获得DC,用反义IDO核酸和AFP基因融合的载体,转染入DC内,检测转染入融合基因DC的对T细胞增殖的影响和体外对肝细胞癌的免疫应答的改变。以~(51)Cr释放法测定细胞毒性T淋巴细胞(CTL)杀伤活性。制作小鼠肝癌模型,分别予以反义IDO-AFP修饰的DC、单纯DC、空白对照组进行治疗,检测血清中自细胞介素(IL)-10、干扰素(IFN)-7水平；CD4、CD8和IFN-7和IL-10双阳性细胞比例,观察其抑制肿瘤的效果。结果反义IDO-AFP修饰的DC可激活小鼠脾淋巴细胞；能对肝癌细胞株H22细胞进行特异性杀伤,杀伤率为89．3%,显著高于单纯DC、生理盐水(空白对照组)组的34．7%和8．9% (P均＜0．01)而对艾氏腹水瘤细胞无效；反义IDO-AFP修饰DC组色氨酸含量为(17．8±1．2)μm,而单纯DC组和空白对照组的色氨酸为(6．2±0．6),um和(5．2±1．2)μm,表明反义IDO-AFP修饰DC可明显增加培养液中的色氨酸。使Thl细胞比例上升,抑制体内肿瘤的生长。结论反义IDO-AFP基因融合修饰树突状细胞后,DC通过调控色氨酸代谢达到高效、特异性抗肝癌的目的,可成为一种有效的瘤苗。     

肝癌细胞裂解物致敏DC疫苗诱导的肿瘤特异抗瘤效应

目的　研究肝癌细胞裂解物致敏的树突状细胞 (DC)疫苗诱导的体内、外抗瘤效应。方法　体外实验 :将肿瘤细胞裂解物致敏的DC与T细胞共培养 ,4d后收获致敏的T细胞 ,观察其对野生型肿瘤细胞的杀伤反应并检测培养液中的IFN γ分泌情况。体内实验 :观察肿瘤细胞裂解物致敏的DC疫苗对小鼠皮下肝癌发生的抑制作用。结果　在效 :靶比分别为 5 0 .0∶1.0、2 5 .0∶1.0、12 .5∶1.0时 ,肝癌细胞裂解物致敏的DC组与DC组诱导的肿瘤杀伤活性分别为 (4 5 .7±3 .2 ) %对 (2 6.5± 2 .5 ) % ;(3 1.0± 2 .7) %对 (14 .3± 3 .3 ) % ;(2 7.8± 1.7) %对 (9.9± 0 .6) % (在各效靶比 ,P <0 .0 1) ,混合细胞培养上清液中IFN γ浓度则为 (2 60 3 .3± 2 60 .0 )ng/L对 (5 0 1.0±5 0 .0 )ng/L。在体内则可有效抑制小鼠皮下肝癌的发生。结论　肝癌细胞裂解物致敏的DC疫苗可以诱导肿瘤特异的抗瘤效应     

mRNA DC疫苗对肝癌细胞增殖周期影响的实验研究

目的 探讨mRNA DC疫苗对肝癌细胞增殖周期的影响,为mRNA疫苗免疫治疗肝癌提供新的实验依据和理论基础.方法 采用粒/巨噬细胞集落刺激因子(GM-CSF)和白介素-4(IL-4)联合培养、扩增鼠骨髓来源DC,以阳离子脂质体转染Hepal-6mRNA入DC,制备mRNA DC疫苗,流式细胞术检测疫苗对体外培养的肝癌细胞周期以及体内种植瘤组织细胞周期的影响;免疫组织化学检测肿瘤细胞增殖核抗原(PCNA).结果 mRNA DC疫苗处理后的肝癌细胞周期比例为G0/G1间期(75.09±3.41)%,S间期(16.93±1.57)%,GO/M间期(7.98±1.83)%,与对照组比较,差别有显著性意义(P＜0.05);荷瘤鼠疫苗组肿瘤细胞增殖指数为(24.91±3.41),与对照组比较,差别有统计学意义(P＜0.05);PCNA实验组阳性表达率为(27.7±10.3)%,与对照组比较,差异有显著性意义(P＜0.05).结论 mRNA DC疫苗可影响肿瘤细胞的分裂周期,减缓细胞的生长,显著抑制肿瘤细胞增殖.     

IL-12基因修饰树突状细胞体外诱导免疫杀伤肝癌细胞

目的探讨IL-12基因修饰树突状细胞(DC)体外诱导免疫杀伤肝癌细胞的效能及其机制。方法IL-12基因修饰肝癌病人外周血DC(DC-IL-12),ELISA法检测DC-IL-12培养上清中IL-12和IFN-γ水平,以冻融HepG2所获得的肿瘤相关抗原(TAA)刺激DC-IL-12,MTT法检测TAA负载的DC-IL-12刺激同源T淋巴细胞增殖分化能力,细胞毒试验检测DC-IL-12诱导的细胞毒T淋巴细胞(CTL)及其上清液对HepG2肝癌细胞株的杀伤作用,ELISA法检测CTL上清液IFN-γ水平。结果DC经IL-12基因修饰后48h分泌高水平IL-12(24·35±5·4)pg/ml及IFN-γ(725±30)pg/ml,均显著高于未转染DC组(P<0·01)。DC-IL-12诱导的CTL及其上清液对HepG2均有显著杀伤作用,杀伤率显著高于未转染DC组,分别为(82·43±8·7)%vs(57·4±4·3)%和(76·45±8·5)%vs(18·23±5·3)%(P<0·01),DC-IL-12诱导的CTL上清液IFN-γ水平显著高于未转染DC组,分别为(1125·0±32·7)pg/ml、(281·3±14·7)pg/ml(P<0·01)。结论IL-12基因修饰增强DC体外诱导自体T淋巴细胞产生特异性抗肝癌免疫,其机制与IL-12基因修饰促进DC分泌IL-12、增强T淋巴细胞活化及分泌IFN-γ密切相关。     

gp96多肽复合物树突状细胞疫苗诱导的体外抗胃癌实验研究

目的研究gp96多肽复合物树突状细胞(DC)疫苗特异性抗胃癌的免疫反应。方法应用层析技术从胃癌组织中提取gp96多肽复合物,制备gp96多肽复合物DC疫苗;采用流式细胞仪检测gp96多肽复合物DC疫苗表面分子的表达;ELISA检测gp96多肽复合物DC疫苗致敏的效应 T淋巴细胞分泌IFN-γ和IL-10的水平;51Cr释放实验检测gp96多肽复合物DC疫苗诱导的特异性对胃癌细胞的杀伤效应。结果gp96多肽复合物DC疫苗表面分子CD1α(79．3±4．1)％、CD80 (84．3±2．4)％、CD83(85．7±3．2)％和HLA-DR(83．4±2．9)％的表达显著增高;对原代培养胃癌细胞的杀伤率(58．5±10．7)％显著高于对SGC 7901细胞的杀伤率(24．0±4．2)％,两者相比差异有统计学意义,P<0．01;对HepG2细胞(13．8±2．8)％则无明显杀伤作用。DC疫苗诱导的效应T淋巴细胞分泌IFN-γ(2875±178)pg／ml的量明显增加,分泌IL-10(36±7)pg／ml的量显著降低。结论 gp96多肽复合物DC疫苗能诱导出较强的对自体胃癌细胞的杀伤作用,且具有高度特异性。     

逆转录病毒携带白细胞介素-12转染树突状细胞体外诱导特异免疫杀伤肝癌细胞

目的探讨逆转录病毒携带白细胞介素(IL)12转染树突状细胞(DC)体外诱导免疫杀伤肝癌细胞的效能及其机制。方法感染指数(MOI)为100,含IL12的重组逆转录病毒修饰肝癌患者外周血DC(DCIL12),酶联免疫吸附试验法(ELISA)检测DCIL12(5×103个/ml)培养上清中IL12和γ干扰素(IFNγ)水平,以冻融肝癌细胞株HepG2(1×107个/ml)所获得的肿瘤相关抗原(TAA)刺激DCIL12,MTT法检测TAA负载的DCIL12刺激同源T淋巴细胞(1×105个/ml)增殖分化能力,细胞毒试验检测DCIL12诱导的细胞毒T淋巴细胞(CTL)及其上清液对HepG2肝癌细胞株杀伤作用,ELISA法检测CTL上清液IFNγ水平。结果DC经IL12基因修饰后48h分泌高水平IL12(24.35±5.40)ng/L及IFNγ(725±30)ng/L,均显著高于未转染DC组(P<0.01及P<0.05)。DCIL12诱导的CTL及其上清液对HepG2均有显著杀伤作用,杀伤率显著高于未转染DC组,分别为(82.43±8.70)%vs(57.4±4.3)%(P<0.01)和(76.45±8.50)%vs(18.23±5.30)%(P<0.01),DCIL12诱导的CTL上清液IFNγ水平显著高于未转染DC组,分别为(1125.0±32.7)ng/Lvs(281.3±14.7)ng/L(P<0.01)。结论IL12基因修饰增强DC体外诱导自体T淋巴细胞产生特异性抗肝癌免疫,其机制与IL12基因修饰促进DC分泌IL12、增强T淋巴细胞活化及分泌IFNγ密切相关。     

肝癌相关成纤维细胞对单核细胞来源树突状细胞分化的影响

目的研究肝癌相关成纤维细胞(hCAF)在单核细胞(Mo)来源树突状细胞(DC)分化过程中所起到的作用。方法采用酶消化法从肝癌组织中分离培养获得人hCAF;采用密度梯度离心法获取健康人外周血单个核细胞(PBMC),经密度梯度离心及磁珠分离法从健康人外周血浓缩白细胞中获得CD14+Mo。hCAF-CD14+Mo(1∶10)和CD14+Mo细胞在第1日及第4日均加入粒细胞-巨噬细胞集落刺激因子(20 ng/ml)和白细胞介素4(20 ng/ml),共诱导6 d。两组细胞分为两份,一半加入脂多糖(LPS)200 ng/ml刺激3 d,另一半不加入LPS作为对照。最后细胞共分为4组:hCAF-Mo组、hCAF-Mo+LPS组、不成熟DC(iDC)组及成熟DC(mDC)组。采用倒置荧光显微镜观察4组细胞的形态变化。各组细胞分为两部分,一部分细胞采用流式细胞术检测细胞表面分子CD83、CD80、CD1a的表达情况,另一部分细胞分别与预染CFSE的淋巴细胞共培养5 d,采用流式细胞术检测T淋巴细胞增殖情况。结果倒置显微镜结果显示在hCAF干扰下Mo不能分化为DC。CD83、CD1a、CD80的表达在iDC组分别为(3.2±0.7)%、(61.7±8.4)%、(30.1±0.9)%,mDC组分别为(80.1±2.8)%、(83.2±6.0)%、(96.1±1.9)%,hCAF-Mo组分别为(1.6±0.9)%、(1.8±0.9)%、(16.0±3.2)%,hCAF-Mo+LPS组分别为(9.0±1.2)%、(1.1±0.4)%、(58.4±3.6)%。hCAF-Mo组与iDC组的CD1a、CD80表达差异均有统计学意义(均为P0.01)。iDC组的CD83表达极低,因此hCAF-Mo组与iDC组的CD83表达差异没有统计学意义(P0.05)。hCAF-Mo+LPS组与mDC组的3种表型表达差异均有统计学意义(均为P0.01)。iDC组的T淋巴细胞增殖率为(3.3±0.9)%,mDC组为(34.5±7.3)%,hCAF-Mo组为(5.3±1.2)%,hCAF-Mo+LPS组为(7.0±1.2)%。与iDC组相比,mDC组能有效地刺激T淋巴细胞增殖。hCAF-Mo组和hCAF-Mo+LPS组刺激T淋巴细胞增殖的能力均较弱,与mDC组相比差异有统计学意义(均为P0.01)。结论 hCAF可明显抑制Mo分化成DC,在肝癌的免疫逃逸过程中发挥重要的作用。     

抑制Tregs增强DC/HCC融合疫苗诱导抗肝癌免疫反应的研究

目的 通过抑制荷瘤小鼠体内调节性T细胞(TregFoxp3+),探讨其增强DC/HCC融合细胞疫苗诱导抗肝癌免疫作用的相关机制.方法 皮下建立小鼠肝癌模型,24只小鼠随机分为4组:(1)环磷酰胺联合疫苗组[环磷酰胺(CTX)+ VAC];(2)疫苗组(VAC);(3)单纯环磷酰胺组(CTX);(4)对照组.各组治疗后,取外周血测定CD8+及CD4+淋巴细胞比例,同时取肿瘤组织及脾脏组织分别行苏木素-伊红(HE)染色和免疫组织化学观察肿瘤坏死和CD8+细胞及Treg+细胞的浸润,并用细胞计数试剂盒(CCK-8)检测脾脏淋巴细胞特异性细胞毒T淋巴细胞(CTL)活性.结果 与其他各组比较,CTX+ VAC组外周血CD4+及CD8+细胞比例增高[ CTX+ VAC:17.50％、14.69％;VAC:16.17％、13.07％;CTX:12.17％、11.59％;对照组:12.24％、11.16％];肿瘤组织可见大量肿瘤细胞坏死,脾脏组织中Foxp3+细胞浸润减少,CD8+细胞浸润增高,CTL对肿瘤细胞杀伤作用明显增强(P＜0.05).结论 通过CTX抑制荷瘤小鼠微环境及外周血中的Treg细胞,可以明显增强DC/HCC融合疫苗对肿瘤的杀伤作用.     

Interstitial cell tumor. Profile of hormone-producing tumor

Interstitial cell tumor of the testis in an adult male can present with feminization and psychologic changes. The feminization and psychologic changes most likely reflect production of estrogen by the interstitial cell tumor. Removal of the tumor causes rapid return of the pituitary-testicular axis to normal, loss of feminization, and development of male psychosocial characteristics.     

Renomedullary interstitial cell tumor

Renomedullary interstitial cell tumor was first introduced by Lerman and co-workers. These lesions have been referred to as medullary fibromas. The ultrastructural studies showed that the spindle cells throughout the stroma have the features of medullary interstitial cells. These benign tumors with specific histology are rare since they are incidental findings at autopsies. We report here a case of renomedullary interstitial cell tumor in a 55-year-old woman.     

Giant cell tumor of bone

Giant cell tumor GCT of bone remains a difficult and challenging management problem because there are no absolute clinical, radiographic, or histologic parameters that accurately predict the tendency of any single lesion to recur or metastasize. Enneking's and Campanacci's radiographic classifications and surgical staging are helpful in planning the initial surgical treatment, because they have observed that a number of the active (Stage 2) lesions and most of the aggressive (Stage 3) lesions have a higher incidence of local recurrence when treated by curettage alone. The bad reputation of curettage and bone grafting is undeserved and arose because of the indiscriminate application of this technique to lesions irrespective of their surgical stage. The ideal aim in the management of GCT is to eradicate the tumor and still save the joint. Curettage, possibly with adjuvant chemical or thermal cauterization, and with bone grafting or polymethyl methacrylate instillation, maintains the structural integrity of the bone and allows for early function. Good results with these techniques when applied to Stage 1 and many Stage 2 lesions may be expected in 70%--80% of the cases. Repetitive freezes with liquid nitrogen, though resulting in a lower recurrence rate, carry with them a not insignificant risk of local complications, require prolonged bracing, and incur the risk of late fracture. When GCTs occur in expendable bones, en bloc resection is the treatment of choice. En bloc resection of major joints requires a facility with reconstruction techniques including the use of allografts, large autogenous grafts and fusion, or custom arthroplasty. These are technically difficult procedures with many early and late complications. Patients have restricted function, and may require prolonged bracing even when uncomplicated. These techniques are therefore reserved for the Stage 3 and selected Stage 2 lesions. Hand lesions have been ineffectively treated by curettage and grafting, and are best treated by early en bloc or ray resection. Multicentric lesions should be handled as individual primary tumors would be in those locations. Radiation therapy has its major role in the treatment of giant cell tumors of the spine and sacrum that are not amenable to complete surgical resection, though long-term sarcomatous change must be looked for. Because of the complex management problem this rare tumor presents, it is recommended that management of giant cell tumor of bone, including the biopsy, the definitive surgery, and the follow-up examination, be carried out by individuals and institutions familiar with this entity.     

Giant cell tumor of bone

This retrospective study analyses 67 patients with 69 giant cell tumors of bone staged according to the Surgical Staging System of benign and malignant lesions of Enneking (1980, 1986). A significant correlation between Stage 2 and Stage 3 benign lesions in respect to local recurrence as well as between the therapeutic procedures and recurrence was found. Intralesional excision with temporary acrylic cement implantation showed to be the preferable initial treatment of these tumors. Special attention to surgical staging, surgical techniques and a supervised rehabilitation program minimize the incidence of recurrence and at the same time giving am maximum of joint function.