转染自体胃癌细胞总RNA的树突状细胞诱导个体化免疫治疗的体外实验

目的探讨转染自体胃癌细胞总RNA的树突状细胞(DC)体外介导抗胃癌的免疫效应。方法制备短期培养的原代胃癌细胞。用rhGM-CSF、rhIL-4和TNF-α体外诱导胃癌患者外周血单个核细胞(PBMC)中DC的发育和成熟,并转染自体肿瘤细胞总RNA,激活自体T细胞产生CTL,用CCK-8试剂盒检测CTL的杀伤活性。应用流式细胞术及混合淋巴细胞培养技术检测DC的免疫功能状态。用ELISA法测定IL-12和INF-γ的水平。结果转染自体肿瘤细胞总RNA的成熟DC,不仅可高表达MHC-I、II类分子及CD80、CD83和CD86协同刺激分子,并可获得高效刺激自体或异体T细胞增殖的能力。转染RNA的成熟DC,分泌IL-12的水平及其刺激产生的CTL培养上清液中INF-γ的水平显著高于单纯成熟DC及未成熟DC;且CTL对自体胃癌细胞的杀伤率显著高于异体组。结论转染自体胃癌细胞总RNA的成熟DC能够体外诱导产生对自体肿瘤细胞具有高度抗原特异性杀伤活性的CTL。     

转染肿瘤总RNA的DC疫苗诱导抗肝癌特异性免疫

目的：探讨转染人肝癌总RNA的树突状细胞(DC) 疫苗体外诱导特异性细胞毒性T淋巴细胞(CTL)的作用。 方法： 采用原发性肝癌（HCC）病人外周血单核细胞（PBMC），在粒/巨细胞集落刺激因子(GM-CSF)和白细胞介素-4(IL-4) 刺激下增殖分化为DC细胞；从人肝癌细胞中体外扩增肝癌RNA。以HCCRNA转染DC细胞，并与PBMC混合培养诱导扩增CTL。MTT法测定CTL的杀瘤活性。 结果： 转染HCCRNA 48 h后， DC表面分子CD83、CD86和HLA-DR表达明显增高。转染HepG-2细胞HCCRNA的DC和病人HCCRNA诱导的CTL对HepG-2细胞和病人HCC细胞的杀瘤活性均明显高于正常肝细胞RNA+DC、脂质体+DC、Opti-MEM+DC以及空白对照组；而对胃癌SGC-7901细胞无杀伤活性。 结论： 以肝癌RNA为肿瘤抗原，DC作为疫苗的抗原提呈细胞，体外冲击致敏DCs，能诱导肝癌特异性CTL。本研究为HCC术后复发和转移的防治提供一种可能有效的疫苗治疗方法。     

肠癌RNA转染小鼠骨髓来源单核细胞的抗肿瘤效应

目的 :以提取的CT 2 6小鼠结肠癌细胞RNA作为抗原物质 ,体外转染骨髓来源的单核细胞 ,回输小鼠体内 ,观察其抗肿瘤效应 ;同时平行比较传统肿瘤冻融抗原体外冲击单核细胞的抗肿瘤效应。方法 :小鼠骨髓细胞体外以GM CSF诱导培养获取单核细胞 ,流式细胞仪检测纯度 ;Trizol法提取获得CT 2 6细胞总RNA ,应用TransMessenger体外转染单核细胞 ,对照组单核细胞以肿瘤冻融抗原冲击 ;LDH释放法检测小鼠体内CTL杀伤活性 ,观察各组小鼠成瘤情况及生存期。结果 :小鼠骨髓细胞经诱导培养后 ,获得大量高纯度的单核细胞 ,流式细胞仪检测CD11b >95 % ;提取的CT 2 6细胞总RNA体外经Trans Messenger介导转染单核细胞后 ,回输小鼠 ,可以诱导体内生成高水平的特异性细胞毒性T淋巴细胞 (CTL)活性 ,使小鼠获得抵抗后继CT2 6细胞攻击的免疫保护能力 ,并显著高于肿瘤冻融抗原冲击的单核细胞回输组。结论 :抗原提呈细胞经肿瘤总RNA转染后 ,可以诱导机体产生CTL ,有效地诱发特异性抗肿瘤免疫功能     

HBsAg重组腺病毒转染的树突状细胞诱导BALB/c小鼠抗HBV免疫的研究

目的 研究HBsAg重组腺病毒转染的小鼠树突状细胞（DC）体内外免疫刺激活性和诱导小鼠抗HBV免疫的特点。方法 BALB／c小鼠的骨髓细胞体外扩增为DC，转染HBsAg重组腺病毒Ad-S或被HBsAg蛋白冲击后，流式细胞术分析DC的表型，混合淋巴细胞反应（MLR）检测DC的刺激活性；或与pcDNA3．1（＋）-S质粒分别免疫小鼠后，LDH法测定脾细胞CTL活性，放免法检测血清抗．HBs；流式细胞术分析体外自体MLR中及免疫后小鼠脾脏T细胞内细胞因子。结果 DC／Ad-S、DC／HBsAg和各对照组DC之间的表型和MLR差异无统计学意义，并均刺激TH和Tc分泌IFN-γ。DC／Ad-S免疫后2周能诱导比DNA疫苗更强产生IFN-γ的TH（P〈0．05），以及比DC／HBsAg和DNA疫苗更强分泌IFN-γ的Tc（均P〈0．01）和特异性CTL（P〈0．05，P〈0．01）。但DC／Ad-S和DC／HBsAg诱导的CTL反应及Tc分泌IFN-γ在免疫后4周均明显减弱，且诱导抗．HBs作用弱于DNA疫苗。结论 HBsAg重组腺病毒转染的DC比HBsAg冲击的DC及DNA疫苗能诱导更强的TH1/Tcl（Ⅰ型）细胞免疫和特异性CTL反应，是诱导抗HBV细胞免疫的有效刺激细胞。     

EBV-LMP2A重组腺病毒体外转染树突状细胞激发特异性CTL的研究

目的：用EBV潜伏膜蛋白2A(EBV-LMP2A)重组腺病毒转染树突状细胞(DC)激发特异性细胞毒性T细胞(CTL)，分析CTL的特性。方法：用AdS-LaMP2A重组腺病毒转染EBV健康携带者及鼻咽癌患者的DC，与自体来源的外周血单个核细胞(PBMC)混合培养，激发LMP2A特异性CIL。用LDH释放法检测CIL杀伤活性；流式细胞术(FACS)检测培养细胞群体中CD3^ 、CD4^ 、CD8^ 、CD56^ 细胞的组成；生物活性法检测细胞培养上清中IFN-γ含量；RT-PCR分析CTL的FasL mRNA表达。结果：EBV健康携带者及鼻咽癌患者的PBMC，经AdS-LMP2A转染的自体DC两次刺激后，都能诱导出显著的EBV-LMP2A特异的CIL。EBV健康携带者CTL的杀伤活性，随DC刺激次数的增加而逐渐增强，诱导的CTL细胞群体以CD^4 和CD8^ 细胞组成为主，且CD4^ 细胞比例高于CD8^ 细胞，另含少量CD56^ 细胞；在不同时段所诱导的CTL上清中，均含一定量的IFN-γ并随刺激次数和诱导时间的延长呈上升趋势；RT-PCR研究表明，所诱导的CTL有FasL mRNA的表达。结论：以腺病毒载体介导EBV-LMP2A基因转染的成熟DC，在体外能激发较强的LMP2A特异的功能性CTL，可用于EBV相关NPC的免疫治疗。     

凋亡癌细胞抗原致敏IL-23基因转染的树突细胞抗肿瘤免疫反应的研究

目的：探讨IL-23基因转染的树突状细胞（DC）负载癌细胞抗原后诱导的免疫应答对小鼠胰腺癌细胞的抑制作用。方法：克隆并构建IL-23基因真核双表达载体，转染DC并负载肿瘤抗原后制备成疫苗。观察各组脾脏T淋巴细胞IFN-γ和IL-4的分泌量以及DC诱导的CTLs对胰腺癌细胞的杀伤作用。结果：基因测序证实IL-23基因克隆及双表达载体构建成功，转染后DC对共刺激分子MHC-Ⅰ和MHC-Ⅱ的表达增强。接种IL-23修饰DC疫苗后小鼠的免疫防御能力显著增强；DC介导的免疫应答促进了IFN-γ生成型Thl细胞的产生，IL-23转染组IFN-γ的分泌与其他各组比较差异显著（P〈0．01）；IL-23转染组T细胞对抑制性细胞因子IL-4的分泌减少，与DC疫苗组和IL-23DC组比较有显著差异（P〈0．05）。转染的DC疫苗在体内诱导出高水平的CTLs活性（P〈0．05）。结论：IL-23使DC抗原递呈能力更强，IL-23修饰DC疫苗可强化宿主针对特异肿瘤的CTLs免疫应答，使宿主不仅产生防御性免疫反应而且增强自动免疫能力。     

CTL识别的HLA-A2限制性人卵巢癌相关抗原OVA66表位的鉴定

目的：鉴定CTL识别的HLA—A2限制性人卵巢癌相关抗原OVA66表位。方法：以细胞因子从外周血单个核细胞(PBMC)中诱导树突状细胞(DC)，通过形态学观察和流式细胞术进行鉴定。用表位预测法选取并合成两种肽分子，分别脉冲成熟的DC，并刺激HLA—A2^ 健康人自体CD8^ T细胞，1wk后，用脉冲肽的自体PBMC以每7d的间隔刺激该CD8^ T细胞3次。以共接受4次抗原肽刺激的T细胞作为CTL，用乳酸脱氢酶(LDH)释放试验，检测CTL对靶细胞的杀伤效应。用酶联免疫斑点法(ELISPOT)．检测CTL中抗原特异性分泌IFN-γ的T细胞数。结果：形态学和流式细胞术的结果显示．PBMC可诱生成熟的DC。肽1235(FLPDHINIV)诱导的CTL．可特异性杀伤1235脉冲的T2细胞和OVA66^ 、HLA—A2^ 的SW480细胞，且L235诱导的特异性分泌IFN-γ的T细胞数增加。结论：卵巢癌相关抗原OVA66的HLA—A2限制性CTL表位1235．能激发对肿瘤抗原的特异性免疫应答，为制备肿瘤特异性肽疫苗奠定了实验基础。     

对携带HCC表位的HBc病毒样颗粒负载的DC疫苗的免疫评价

目的:探讨携带肝细胞癌(HCC)表位的嵌合蛋白颗粒HBc-VLPs负载小鼠BMDCs制备的DC疫苗,免疫小鼠后诱导抗原特异的细胞免疫应答和抗肿瘤效应。方法:制备HLA-A2转基因小鼠来源的BMDCs,将三种带有高表达于HCC的HLA-A2限制的CTL表位的HBc-VLPs、三种抗原表位肽和不含HCC表位野生HBc-VLPs分别负载HLA-A2转基因小鼠BMDCs制备成DC疫苗,阴性对照组用PBS替代。将HLA-A2转基因小鼠分为四组,分别免疫上述DC疫苗并对其免疫效果进行评价:体外检测抗原特异性淋巴细胞内IFN-γ的产生和CTL细胞活性并观察不同方法处理的DCs对肿瘤生长的抑制情况。结果:与其他组相比,HBc-VLPs负载的DCs免疫小鼠后第8天,流式细胞仪即可检测到分泌IFN-γ的CD8+T细胞存在;用LDH法检测到较强的抗原特异性CTL活性;在肿瘤接种后的20天之前均未见到明显的肿瘤块,具有显著抗肿瘤作用。结论:利用携带HCC抗原表位的HBc-VLPs负载DCs后得到可强烈诱导免疫活性和明显抗肿瘤作用的DC疫苗,为DC疫苗的优化和评价提供了实验方法,也为进一步深入研究新型肝细胞癌治疗性疫苗的功能和应用奠定了基础。     

ΔNp73α基因转染树突状细胞抗乳腺癌细胞的免疫效应

目的:研究ΔNp73α基因转染树突状细胞(DC)诱导的特异性抗乳腺癌免疫效应。方法:人脐带血细胞经GM-CSF、IL-4、TNF-α等细胞因子诱导培养DC,流式细胞仪(FCM)检测DC成熟前后CD1a、CD83的表达变化情况。脂质体法将pc DNA-HA/ΔNp73α转染至DC,经Western blot检测转染情况。转染DC与自体T细胞共培养诱导特异性CTL。MTT法测定T细胞增殖能力;ELISA法检测IFN-γ的分泌水平;LDH释放法检测T细胞对乳腺癌细胞MDA-MB-231的杀伤作用。结果:DC诱导成熟后,CD1a表达约占56%,CD83约占74%,与未成熟DC(CD1a 19%,CD83 13%)比较,差异有显著统计学意义(P0.01)。Western blot检测到DC-ΔNp73α组有一特异条带表达。DC-ΔNp73α组诱导的特异性CTL对MDA-MB-231杀伤作用高于DC组(P0.05),而且刺激T细胞增殖能力增强,分泌IFN-γ的水平升高,与空载体DC-pc DNA组及DC组比较有显著统计学意义(P0.01)。结论:以ΔNp73α转染DC制备的DC疫苗,具有显著诱导CTL杀伤乳腺癌细胞的作用。     

树突细胞对Lewis肺癌抗瘤作用的研究

目的：研究经体外骨髓细胞衍生的DC在Lewis肺癌中的抗瘤作用。方法：将肿瘤抗原MUT－1加载的DC（DC－MUT－1）经尾静脉免疫C57BL／6小鼠后，随后将肿瘤细胞接于小鼠皮下，观察DC能否诱发机体的免疫保护。同时检测DC－MUT－1免疫后小鼠脾脏T细胞的CTL活性。结果：经DC－MUT－1免疫后能诱发机体产生对相应肿瘤细胞的持久和特异性的免疫保护作用，体外检测CTL活性显示，免疫小鼠脾脏T细胞对相应肿瘤具有 特异性杀伤。结论：经DC－MUT－1免疫小鼠能诱发机体产生特异性抗肿瘤细胞免疫从而使宿主对随后的肿瘤细胞攻击产生特异性的免疫保护作用。     

Clear cell basal cell carcinoma

We describe a case of clear cell basal cell carcinoma of the superficial type, presenting as a crusted eruption on the abdomen. Histological examination showed a solid proliferation of clear cells attached to the under-surface of an atrophied epidermis. In addition, distinct pagetoid infiltration was seen within the overlying epidermis. A focal connection between the clear cell portion and a deeper lying nodular basal cell carcinoma was demonstrated, elucidating the true nature of the lesion. Immunohistochemical studies and electronmicroscopy confirmed the epithelial derivation of the tumour. The clear cell appearance was due to multiple cytoplasmic electronlucent vacuoles which were not surrounded by membranes.     

睾丸大细胞钙化性支持细胞瘤一例

患者男,12岁。左侧睾丸无痛性肿大1年余,于2005年月18日入院,右侧睾丸未见异常,亦无第二性征的异常。病理检查:送检组织为带一段精索和附睾组织的睾丸及其肿物。精索长5.0cm,直径1.2cm;附睾大小3.0cm×1.5cm×0.8cm,表面未见异常;睾丸及肿物大小3.0cm×2.5cm×2.5cm,表面光滑,包膜完整。切面实性,灰黄色,质硬,有钙化,无出血和坏死。肿物几乎占据整个睾丸实质,肿瘤周围边界尚清楚。镜下检查:肿瘤组织与周围边界清图1睾丸大细胞钙化性支持细胞瘤,肿瘤细胞圆形、卵圆形,含有丰富的润,间质黏液样变性HE中倍放大图2肿瘤组织间质中可见大量呈棕褐…     

Chondrocytic cell differentiation in clear cell chondrosarcoma

Clear cell chondrosarcoma is a rare mesenchymal neoplasm of unclear differentiation. Besides having a chondrogenic nature, an osteogenic differentiation was also proposed. In this study, expression analysis of extracellular matrix genes, which are specific for different mesenchymal cell differentiation pathways, were used to get a better understanding of origin and differentiation pattern of the clear cell chondrosarcoma tumor cells. Our in situ analysis of two cases shows that (1) chondrocytic cell differentiation as marked by the expression of cartilage collagen type II and proteoglycans is a characteristic feature within the development of the neoplasm, (2) multifocal chondrocyte hypertrophy as shown by the expression of type X collagen does occur, and (3) no significant expression cf collagen type I, the main gene product of osteoblastic cells, is found by the neoplastic cells. Thus, our study indicates that clear cell chondrosarcoma shows a chondrogenic, but not osteogenic, differentiation and represents a true chondrosarcoma. The unusual scarcity of its extracellular and the multifocal expression of type X collagen marks clear cell chondrosarcoma as a chondrosarcoma tumor entity of a particular cell differentiation pattern. The expression of cartilage type collagens represents a distinct marker from bone metastases of clear cell neoplasms of other origins.     

Stem cell evolutionary paradigm and cell engineering

Studying hematopoietic and mesenchymal stem cells for almost three decades revealed some similarities between the stem cell entity and the single-celled eukaryotes exhibiting the anaerobic/facultative aerobic metabolic features. A careful analysis of nowadays knowledge concerning the early eukaryotic evolution allowed us to reveal some analogies between stem cells in the metazoan tissues and the single-celled eukaryotes which existed during the first phase of eukaryotes evolution in mid-Proterozoic era. In fact, it is possible to trace the principle of the self-renewal back to the first eukaryotic common ancestor, the first undifferentiated nucleated cell possessing the primitive, mostly anaerobically-respiring mitochondria and a capacity to reproduction by a simple cell division “à l’identique”. Similarly, the diversification of these single-cell eukaryotes and acquiring of complex life cycle allowed/conditioned by the increase of O₂ in atmosphere (and consequently in the water environment) represents a prototype for the phenomenon of commitment/differentiation. This point of view allowed to predict the ex-vivo behavior of stem cells with respect to the O₂ availability and metabolic profile which enabled to conceive the successful protocols of stem cell expansion and ex vivo conditioning based on “respecting” this relationship between the anaerobiosis and stemness. In this review, the basic elements of this paradigm and a possible application in cell engineering were discussed.     

Mutagensis and cell transformation in cell culture

The current lack of continuous prawn cell lines suitable for the isolation and growth of prawn viruses is a major setback for diagnosis of viral diseases of prawns; isolation and identification of the causative agents are severely hindered and the development of other diagnostic procedures is slowed. To date, there are few, if any, examples of continuous prawn cell lines suitable for virus isolation and growth. Recent studies indicate that clastogenic agents such as ionising radiation are more effective than powerful point mutagens as immortalising agents for mammalian cells. Such studies with mammalian cell cultures indicate that certain mutagenic agents and/or prolonged treatment with a combination of mutagens may prove more useful in the production of immortal prawn cell lines in vitro rather than other, more limited and specific procedures. This report provides a brief review of the use of mutagens to induce immortalisation -- the acquisition of unlimited growth potential -- in mammalian cells in culture, i.e. the production of continuous cell lines from primary cultures. Aspects of cell transformation vs. immortalisation, the control of cell growth in vitro, and the application of mutagenic agents to induce immortalisation in primary cultures of prawn tissues will be discussed.