骨髓间充质干细胞对异体T细胞几种细胞因子分泌的影响

目的:探讨骨髓间充质干细胞(MSC)对异体T淋巴细胞分泌IL-2、IL-4、IL-10、IFN-γ的影响,从而进一步探讨MSC诱导免疫耐受的机制。方法:将培养了3代后的MSC经照射后作为基底层细胞,接种分离的异体T淋巴细胞。共培养7天后,用ELASA方法检测上清中的上述四种细胞因子的蛋白含量,同时计数T淋巴细胞数量的变化,应用流式细胞仪检测与MSC共孵育前后T淋巴细胞CD3、CD4、CD8、CD25的变化。结果:(1)ELASA检测IFN-γ分泌量为667·07±14·76pg/ml;IL-10为46·19±1·41pg/ml;IL-2为33·01±5·92pg/ml;IL-4为0·15pg/ml。(2)共孵育组T淋巴细胞在数量上明显减少。(3)CD8+细胞数增多,CD25+细胞减少。结论:骨髓MSC在体外与异体T淋巴细胞共孵育后IFN-γ高分泌,可能与共孵育后T淋巴细胞亚群的改变有关。这一研究结果为预防HLA不相合的骨髓移植的GVHD的发生提供了一条新的思路。     

消化道肿瘤浸润淋巴细胞的提取、培养及表型分析

用机械分离、酶解聚和非连续密度梯度离心方法从28例消化道肿瘤制取肿瘤浸润淋巴细胞(TIL)单细胞悬液。每克湿重瘤组织细胞得率为20.78±9.82×10~6(6.09～78.40×10~6),淋巴细胞占67.29±11.78％。相同方法自17例肿瘤区域淋巴结中得到的细胞为38.08±17.42×10~6/每克淋巴结,淋巴细胞占82.08±8.48％。在含高浓度rIL-2中培养时、区域淋巴结淋巴细胞(RNL)增殖力最强,PBL的增殖反应发生最早,但扩增时间短。免疫荧光标记流式细胞仪分析,患者淋巴细胞CD4比例减少,CD8增加,CD4/CD8显著降低,CD25~+细胞增加。经rIL-2培养后,T细胞(CD2~+,CD3~+)增加,多数样本以CD8生长为主,对照组以CD4生长为主。CD25~+细胞逐渐增加。TIL、RNL、PBL中Tac抗原最高表达率分别达36.8％、44.2％、20.6％。     

生长抑素对猕猴肠缺血再灌注后回肠黏膜IFN-γ的影响

探讨生长抑素(somatostatin,SST)对肠缺血再灌注(intestinal ischemia reperfusion injury,IIR)后回肠黏膜IFN-γ产生及IIR免疫炎症损伤的影响。取15只健康猕猴分为正常对照、IIR、SST+IIR三组(n=5),ELISA检测回肠组织、肠上皮细胞、肠淋巴细胞、肠淋巴细胞孵育上清液、外周血、门静脉血中IFN-γ的水平;免疫组化检测IFN-γ、CD4、CD8、CD57的分布,图像分析软件测定阳性面积及IOD值。发现,IIR后回肠黏膜上皮细胞、门静脉血内IFN-γ水平较正常组显著增加[分别为(30.7±20.1)pg/mg和(9.4±2.4)pg/mg总蛋白;(18.1±1.1)pg/ml和(8.7±1.3)pg/ml](P<0.05),而肠淋巴细胞及其上清液的IFN-γ水平较正常组明显降低[分别为(1.7±0.02)pg/mg和(11.1±4.0)pg/mg总蛋白;(2.2±0.3)pg/mg和(5.2±1.5)pg/mg](P<0.05);预防性给予SST后,与IIR组比较,回肠上皮细胞及门静脉血内IFN-γ的水平显著下降[分别为(13.6±4.9)pg/mg总蛋白,P=0.055;(8.9±2.5)pg/ml,P<0.05],肠淋巴细胞及其上清液的IFN-γ水平无明显变化。组织匀浆及外周血的IFN-γ水平在三组间无明显差别。CD4+细胞、CD57+细胞分布与IFN-γ无关,CD8+细胞分布与IFN-γ变化趋势相反。因之,SST负性调控IIR时回肠上皮细胞IFN-γ的产生,从而可能阻止多器官功能障碍的发生发展。     

肿瘤微环境中IL-10限制SOCS1基因沉默的树突状细胞疫苗的抗肿瘤作用

目的观察SOCS1基因沉默的DC疫苗对荷黑素瘤小鼠的抗肿瘤作用及肿瘤微环境中IL-10对该DC疫苗抗瘤作用的影响。方法从小鼠股骨分离骨髓细胞,GM-CSF和IL-4联合诱导DCs分化,然后感染携带沉默SOCS1基因的Len-SOCS1-shRNA慢病毒;用TRP2抗原肽负载沉默SOCS1的DC细胞制备DC疫苗,LPS诱导成熟,流式细胞仪分析DC细胞表面MHCⅡ和CD86的表达,real-time PCR分析该DC细胞的SOCS1、IL-10和IL-12p40表达。用B16细胞或降低IL-10基因表达的B16(B16-IL-10-/-)制备荷瘤小鼠模型,瘤内注射DC疫苗(3×106/只),观察肿瘤生长和荷瘤小鼠生存期。采用不连续梯度离心法分离肿瘤浸润淋巴细胞,流式细胞仪观察CD8+T细胞的分布;并采用微量细胞毒的方法分析CTL活性。结果 Len-SOCS1-shRNA慢病毒感染DC后,可使SOCS1表达下调80%;下调SOCS1表达的DC细胞MHCⅡ和CD86表达有增加趋势,但与对照组DC相比无明显差异;下调SOCS1表达可降低DC细胞的IL-10表达,提高IL-12p40的表达。沉默SOCS1的DC疫苗对B16荷瘤小鼠的生存率没有明显影响,但可显著提高B16-IL-10-/-荷瘤小鼠的生存率(P<0.05)。下调SOCS1表达的DC疫苗不仅可提高B16-IL-10-/-荷瘤小鼠肿瘤浸润CD8+T淋巴细胞数,还可促进CD8+T细胞IFN-γ的分泌及CTL活性。结论沉默SOCS1可提高DC疫苗的活性,但肿瘤微环境的IL-10依然是限制该DC疫苗有效发挥抗瘤作用的因素。     

骨关节炎与免疫关联性初探

为了探讨免疫因素是否介导骨关节炎的发病 ,应用直接免疫荧光法检测了 6例晚期骨关节炎患者滑膜液中浸润淋巴细胞分化抗原的表达 ,同时用微量酶联免疫技术测定了这些患者滑膜液中IL 10、IL 12和sFasL的含量。结果显示关节滑膜液中的淋巴细胞以CD8+ 细胞为主 (6 7 0 %± 14 6 %) ,远远高于CD4+ 细胞 (15 0 %± 12 8%) ,两者相差显著 (P <0 0 1)。CD4+ /CD8+ 比值 <1。进一步分析表明其T细胞受体主要为αβ型TCR (6 4 0 %± 12 6 %)。滑膜液中IL 12含量 (2 0 3 84±49 2 )pg/ml高于IL 10 (37 8± 14 5 )pg/ml,有明显统计学差异 (P <0 0 1)。外周血中IL 10和IL 12的含量分别为 (4 3 37±14 2 )pg/ml和 (4 5 5 8± 10 6 )pg/ml。血清中sFasL含量较高 (15 3 90± 5 6 )pg/ml,而滑膜液中则很低 (<5pg/ml,P <0 0 1)。提示在骨关节炎的晚期CD8+ 淋巴细胞和IL 12增高可能参与了关节的免疫损伤。     

肿瘤及肿瘤化疗病人外周血单个核细胞诱生γ—干扰素的比较

本文用高亲和力、高特异性的人重组γ-干扰素(rhIFN-γ)单克隆抗体A_3,建立的人γ-干扰素的免疫放射量度分析。检测范围为0.10～250U/ml.批内和批间变异系数分别为7.40%和17.3%,回收率为1±0.17,稀释试验符合线性关系(r=0.98),不与IFN-α,IFN-β交叉。用本法检测了29例正常人、34例肿瘤病人和19例肿瘤化疗病人外周血单个核细胞诱生IFN-γ的量。结果表明肿瘤病人和正常人外周血单个核细胞产生IFN-γ的能力无明显差别,化疗病人外周血单个核细胞产生IFN-γ能力显著低于正常人及未化疗的肿瘤病人(P<0.005),这可能是由于化疗杀伤了CD_4~+、CD_(?)~+细胞。     

类风关滑膜浸润T细胞特性与致病机制研究

为研究类风湿关节炎时关节滑膜浸润性T细胞生物学特性与致病机制 ,对 10例RA患者滑膜液中淋巴细胞的免疫表型、细胞因子分泌格局与趋化因子受体表达进行了分析。用双色荧光标记法分别测定滑膜液中和外周血淋巴细胞表型与趋化因子受体表达。用ELISA方法检测滑膜液与外周血中IFN γ、IL 10、IL 4与IL 12的含量。结果是滑膜液中的CD4 + T淋巴细胞为 4 0 0 %± 11% ,CD8+ T细胞为 34 0 %± 6 % ,CD4 + 与CD8+ T细胞的比值为 1 2 ,显著低于外周血中CD4 /CD8的比值。滑膜液中CD3和CD2 5双阳性的活化T细胞占 16 %± 6 0 %。趋化因子受体CCR5表达较低 ,与外周血无明显差异。但CX CR3表达水平较高 ,为 16 %± 4 0 % ,远远高于外周血 (仅为 0 5 %± 0 3% )。IFN γ在滑膜液中含量很高 ,达 (36 6 7± 4 3 2 )pg/ml,而外周血中含量仅为 (2 0 1± 3 2 )pg/ml。IL 4含量未能测得 (<15pg/ml ) ,与外周血相似。IL 12含量为 (4 19 9±89 2 )pg/ml,远高于外周血中的含量 (6 5 32± 34 2 )pg/ml。IL 10含量为 (187 7± 34 5 )pg/ml,高于外周血中的含量 (85±12 7)pg/ml。在所测细胞因子中 ,关节滑膜液中IFN γ和IL 12的含量与外周血相比具有显著的统计学差异。表明RA关节滑膜液中有相当数量的T细胞浸润。这些T细胞     

腺病毒编码人TRP2修饰DC诱发抗小鼠黑色素瘤免疫的研究

目的比较腺病毒载体(Ad)介导人和小鼠酪氨酸酶相关蛋白2(tyrosinase-relatedpro-tein2,TRP2)修饰小鼠骨髓来源的树突状细胞(BM-DC)诱发抗小鼠黑色素瘤免疫的差异。方法Ad编码人或小鼠TRP2(AdhTRP2或AdmTRP2)体外感染小鼠BM-DC并体内皮下免疫C57BL/6小鼠,7d后取出被免疫小鼠脾细胞行体内细胞毒性T淋巴细胞杀伤试验(invivoCTL)和细胞内IFN-γ染色(ICS)分析CTL的杀伤活性和IFN-γ的产生;或给免疫后小鼠皮下接种小鼠B16.F10黑色素瘤细胞,观察荷瘤小鼠的成活情况。结果invivoCTL和ICS分析显示,AdhTRP2/BM-DC免疫小鼠,其6hCTL杀伤率为(98.7±1.2)%,IFN-γ产生的CD8+T细胞占总CD8+T细胞的(1.25±0.21)%;而AdmTRP2/BM-DC免疫的小鼠,其6hCTL杀伤率和产生IFN-γ的CD8+T细胞比例分别为(28.6±6.3)%和(0.24±0.06)%。荷瘤试验表明,AdhTRP2/BM-DC免疫小鼠后1周皮下接种106B16.F10细胞,观察3个月100%的小鼠无瘤生长;而接种5×104B16.F10细胞至AdmTRP2/BM-DC免疫1周的小鼠,3个月后小鼠成活率仅为40%。结论Ad介导异种(人)TRP2较自身(小鼠)TRP2修饰的BM-DC更为有效地打破肿瘤免疫耐受、诱导强烈的抗黑色素瘤免疫反应,是一种高效的以DC为基础的肿瘤疫苗。     

胃癌细胞总RNA修饰树突状细胞的肿瘤疫苗效果

目的应用肿瘤细胞RNA修饰小鼠树突状细胞(dendrticcell,DC)的肿瘤疫苗免疫小鼠,研究其抗肿瘤免疫作用.方法CD11c磁珠抗体标记小鼠脾单个核细胞悬液,磁性细胞分选器(MACS)分选出CD11c+的DC,短期培养,以肿瘤细胞总RNA脉冲DC,以此瘤苗皮下注射免疫615小鼠两次,间隔1周,最后1次免疫后7d,实验鼠皮下接种5×105小鼠前胃癌细胞(MFC).结果经免疫后的小鼠具有较强抗肿瘤免疫,RNA脉冲DC的瘤苗免疫后肿瘤生长受到明显抑制,其外周血和脾细胞中NK活性均明显升高,肿瘤特异性CTL也明显升高.结论以肿瘤RNA作为抗原脉冲DC可诱导较强的抗肿瘤免疫.     

体外大量扩增原发性肝癌肿瘤浸润淋巴细胞及其生物学特性的初步分析

目的探讨体外大量培养扩增原发性肝癌肿瘤浸润淋巴细胞(TILs)的新方法,并初步研究其分子表型和功能特征。方法 20例手术切除的原发性肝癌组织经混合消化酶消化为单个细胞后,应用密度梯度离心法分离其中的淋巴细胞,然后应用两步培养法大量扩增TILs,并检测它们的生长扩增速度。应用流式细胞仪检测扩增淋巴细胞的分子表型,ELISA法检测其IFN-γ的分泌水平,并分析高表达PD-1和低表达PD-1分子TILs分泌IFN-γ的能力。结果 18例标本中的TILs在体外经两步法培养后可以扩增达到1010数量级。扩增的TILs中CD3~+细胞的比例为(91.7±7.4)%,CD3~+CD56~+细胞的比例为(28.5±11.7)%,CD3~+CD4~+细胞的比例为(32.5±19.6)%,CD3~+CD8~+细胞的比例为(62.5±29.1)%。PD-1分子在CD3~+CD8~+细胞的比例为(23.5±16.1)%。扩增的TILs分泌INF-γ的水平为(827.8±307.7)pg/ml,其中9例PD-1分子低表达的CD3~+CD8~+细胞的分泌INF-γ的水平为(1087.5±249.6)pg/ml,6例PD-1分子高表达的CD3~+CD8~+细胞的分泌INF-γ的水平为(478.6±69.3)pg/ml,两者相比有明显差异(P0.05)。结论应用两步培养法从原发性肝癌中大量扩增TILs简便易行,扩增的TILs主要为CD3~+CD8~+T淋巴细胞,且TILs中PD-1的表达与其分泌IFN-γ的能力呈负相关。     

Spinal pleomorphic xanthoastrocytoma companied with periventricular tumor

Pleomorphic xanthoastrocytoma (PXA) is a low grade tumor that occurs in supratentorial area of children and young adult. In the previous reports, PXA of spinal cord or multicentre was extremely rare. A 60-year-old patient of spinal PXA and periventricular tumor presented with waist pain and weakness of double legs for one month. Neuroimaging showed that a lesion at the level of L2-L3 and periventricular tumor. Postoperative microscopy indicated that WHO grade II PXA. Photomicrograph of the lesion showed spindle cells, marked nuclear and cytoplasmic pleomorphism, with foamy cytoplasm. Immunohistochemical staining showed that GFAP and S-100 were positive. This is a rare case of synchronous multicentric PXA. Physicians should be realized multicentric dissemination by meninges or cerebrospinal fluid in PXA patients. It is important to describe the particular case in order to better understanding of clinical features.     

Increased production of tumor necrosis factor during endotoxin shock in mice presensitized with serum of mice with tumors or with tumor cell factors

Laboratory of Cellular Immunopathology and Biotechnology, Research Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow (Presented by Academician of the Academy of Medical Sciences of the USSR N. K. Permyakov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 7, pp. 78–80, July, 1991.     

Thymoma with ganglioneuroblastomatous component: case report

A mediastinal tumor in a 49-year-old woman with myasthenia gravis is reported. The tumor was well-demarcated and located in the supero-anterior mediastinum. Microscopically, the tumor consisted of thymic and neuroblastic tumor components, the latter of which consisted of immature and maturing neuronal cells, abundant neuropils, and Schwannian stroma. The two components intermingled with each other inside the tumor capsule. The tumor was diagnosed as thymoma with a ganglioneuroblastomatous component. The coexistence of epithelial and neuronal tissues in the thymic neoplasm is extremely rare.     

Epithelioid angiomyolipoma of kidney with atypical nuclear features and intranuclear inclusions on cytology

Described herein are the cytological findings of epithelioid angiomyolipoma (EAML) of the kidney with atypical nuclear features mistaken for renal cell carcinoma (RCC) in a 61‐year‐old male patient. Aspirates from this large renal mass were cellular and showed epithelioid cell clusters with focally crowded nuclei showing moderate anisonucleosis, small nucleoli, and prominent eosinophilic intranuclear inclusions. Failure to recognize the scanty adipose tissue component and preponderance of epithelioid cells with nuclear pleomorphism lead to a diagnosis of RCC on cytology. On histology, the tumor was essentially composed of epithelioid and spindle cells that showed the typical immunoprofile of an angiomyolipoma and only occasional foci of typical AML were seen. The hilar lymph node was involved in contiguity. However, in view of lack of obvious features of malignancy, the tumor was labeled as EAML with atypical features. Immunocytochemistry on the destained cytology aspirates revealed strong smooth muscle actin staining of all cells. To conclude, EAML can mimic a RCC. In such instances, lack of arborizing vasculature, absence of cytoplasmic fatty vacoulation, crowded nuclei with intranuclear inclusions, and lack of prominent nucleoli along with typical immunophenotype of EAML may assist in the cytology diagnosis. Diagn. Cytopathol. 2011;39:278–282. © 2010 Wiley‐Liss, Inc.     

Collision tumor of the stomach: Gastric adenocarcinoma intermixed with gastrointestinal stromal tumor

The synchronous occurrence of tumors of different histological types in the stomach is uncommon. This rare association is usually detected incidentally at gastroscopy, surgery or histopathology. It is still not known whether such association represents an incidental coexistence or indicates a similar pathogenesis in the simultaneous development of tumors of different histological types in the same organ. A 73‐year‐old woman complained of discomfort after eating a Chinese dumpling, and had tarry stools three times. Gastroscopy showed a tumor at the juncture of the gastric fundus and corpus. The tumor was composed of gastric adenocarcinoma and gastrointestinal stromal tumor, in which the two components were intermixed with each other. To the authors' knowledge there are few reports of this extremely rare collision tumor association.     

Multiple small intestinal stromal tumors with skeinoid fibers in association with neurofibromatosis 1 (von Recklinghausen's disease)

A case of multiple small Intestinal stromal tumors (SIST) with skeinoid fibers of the jejunum arising in a 50 year old male with neurofibromatosis 1 (NF-1) is reported. Seven small tumors of the jejunal wall were incidentally found and excised during an operation for abdominal and retroperitoneal neuroflbromas. Histologlcally, the tumors were composed of uniform spindle-shaped cells with fascicular pattern, almost indistinguishable from the histology in leiomyoma. Periodic acid Schiff stain-positive hyaline globules were observed among the tumor cells. Ultrastructurally, these globules were stromal tangles of curvilinear, fluffy fibrils, consistent with skeinoid fibers. The electron-dense granules, possibly neurosecretory granules, were found In the cytoplasm of the tumor cells. Immunohistochemically, the tumor cells were positive for vimentin, neuron specific enolase and CD34, but negative for muscle markers and S100 protein. The association of NF-1 and multiple SIST with skeinoid fibers may have clinical implications. The multiple occurrence of SIST with skeinoid fibers seems to be often cited as one of the gastrointestinal manifestations of NF-1. The possible site of origin of SIST with skeinoid fibers in NF-1 may be the enteric autonomlc nerve plexus in the small intestinal wall.     

Pineal malignant rhabdoid tumor with chondroid formation in an adult

A pineal tumour in a 27-year-old male is presented with the characteristic histological features of a pineal malignant rhabdoid tumor (MRT) with chondroid formation. Occasionally, tumor cells contained a single well-demarcated hyaline globular inclusion within the cytoplasm adjacent to the nucleus. The stroma of these tumors tends to be densely hyalinized and become chondroid. Immunohistochemical staining was positive for vimentin, epithelial membrane antigen, chromogranin A, synaptophysin, neuron-specific enolase, S-100 protein, and muscle actin. Despite surgery and radiochemotherapy, the tumor recurred in the pineal region and metastasized to the lower lobe of right lung. The patient died 2 years after the initial diagnosis. This is the second published case of central nervous system-MRT appearing in an adult. The clinical and pathological features of pineal MRT in this patient are presented.     

A Testicular Teratoma with Rhabdomyosarcoma and Seminoma

A case of testicular mixed germ cell tumor with teratoma in a 36-year-old Japanese male is reported. Histologically, it was strongly suggested that the rhabdomyosarcoma had originated from the mesenchymal element of the teratoma. A review of the literature revealed six definitive and two possible previous cases of testicular rhabdomyosarcoma with teratoma, either with or without other germ cell malignancies. However, the present case associated with seminoma as a germ cell tumor is the first of its kind to be reported. In contrast to the poor prognosis observed in the previous cases, the present patient remains free of disease 3 years and 3 months after orchiectomy, chemotherapy and irradiation. Acta Pathol Jpn 41: 707–711, 1991.     

Cytology of sex cord tumor with annular tubules: A case report

The cytologic findings of a sex cord tumor with annular tubules (SCTAT) that ruptured during laparoscopy are described. Features useful in distinguishing SCTAT from other ovarian sex cord tumors include the presence of highly cellular tubular formations containing well-delimited glassy pink material and absence of single cells. To the best of our knowledge, the cytology of SCTAT has not been previously reported. Diagn. Cytopathol. 1998;18:362–364. © 1998 Wiley-Liss, Inc.     

A study of early pregnancy factor activity in the sera of women with trophoblastic tumor.

PROBLEM: To detect whether or not the early pregnancy factor (EPF)-like activity, or chaperonin 10, could be in the sera of patients with trophoblastic tumor in order to find another more efficient means to diagnose this kind of tumor. METHOD OF STUDY: The rosette inhibition assay was used to detect EPF-like activity in 216 sera, collected from patients with gestational trophoblastic tumor, including 47 sera of patients with choriocarcinoma, 68 sera of patients bearing invasive mole, and 101 sera of patients with vesicular mole. RESULTS: The accuracy of diagnosing malignant trophoblastic tumor by detecting EPF-like activity is 91.3% (105/115), with a false positive rate of 14.58% and a false negative rate of 4.8% by this method. Furthermore, the rosette inhibition titer (RIT) values have significant difference (P < 0.001) between the sera in patients with malignant trophoblastic tumor before treatment and those after treatment. CONCLUSIONS: This study demonstrated that diagnosis of malignant trophoblastic tumor could be made with an accuracy of 91.3% by detecting EPF-like activity and that EPF-like activity could be used as an indicator to distinguish benign from malignant trophoblastic tumor.