CARP相互作用蛋白HTRM的表达谱分析及亚细胞定位

目的观察CARP相互作用蛋白HTRM的组织表达谱和亚细胞定位及其对血管平滑肌细胞（VSMC）增殖的作用。方法我们利用CLONTECH公司的成人多组织膜，通过Northern杂交的方法，检测HTRM在心脏、脑、胎盘、肺、肝、骨骼肌、肾脏以及胰腺中的表达情况。将HTRM带上GFP标签转染HEK293S细胞，观察其亚细胞定位情况。再将HTRM及对照载体转染VSMC细胞，通过细胞计数以及MTr两种方法检测细胞的增殖情况。结果NorthernBlot实验显示HTRM有两个转录本，2．4kb的转录本主要在心脏和骨骼肌中表达，1．35kb的转录本主要在胎盘中表达；亚细胞定位显示HTRM是一个胞浆蛋白；高表达HTRM可以轻微的促进VSMC细胞增殖。     

Neurologic Autoimmunity in the Era of Checkpoint Inhibitor Cancer Immunotherapy

Neurologic autoimmune disorders in the context of systemic cancer reflect antitumor immune responses against onconeural proteins that are autoantigens in the nervous system. These responses observe basic principles of cancer immunity and are highly pertinent to oncological practice since the introduction of immune checkpoint inhibitor cancer therapy. The patient’s autoantibody profile is consistent with the antigenic composition of the underlying malignancy. A major determinant of the pathogenic outcome is the anatomic and subcellular location of the autoantigen. IgGs targeting plasma membrane proteins (eg, muscle acetylcholine receptor -IgG in patients with paraneoplastic myasthenia gravis) have pathogenic potential. However, IgGs specific for intracellular antigens (eg, antineuronal nuclear antibody 1 [anti-Hu] associated with sensory neuronopathy and small cell lung cancer) are surrogate markers for CD8⁺ T lymphocytes targeting peptides derived from nuclear or cytoplasmic proteins. In an inflammatory milieu, those peptides translocate to neural plasma membranes as major histocompatibility complex class I protein complexes. Paraneoplastic neurologic autoimmunity can affect any level of the neuraxis and may be mistaken for cancer progression. Importantly, these disorders generally respond favorably to early-initiated immunotherapy and cancer treatment. Small cell lung cancer and thymoma are commonly associated with neurologic autoimmunity, but in the context of checkpoint inhibitor therapy, other malignancy associations are increasingly recognized.     

CARP编码蛋白的相互作用蛋白筛选

目的 :筛选与CARP编码蛋白相互作用的蛋白 ,为其功能研究奠定基础。方法 :将编码全长CARP的DNA序列插入到pGBKT7 BD载体中 ,转化AH1 0 9酵母 ,然后与含有已克隆到pACT2载体上的人心脏cDNA文库的Y1 87酵母融合。CARP与相应的人心脏cDNA片段编码的蛋白发生相互作用后 ,可激活报告基因的表达。阳性克隆的质粒进行测序分析。同源性检索搜寻GenBank中与之相同或相似的序列。结果 :共筛选约 3× 1 0 7个cDNA ,筛选出 9个阳性克隆 ,其中包括FLNa (actin bindingprotein 2 80 )。结论 :CARP编码蛋白在酵母中可以特异性的结合FLNa ,提示CARP可能通过与FLNa相互作用参与调控细胞增殖     

CARP基因重组腺病毒载体的构建

目的：利用Adeasy-1系统，构建并鉴定CARP基因重组腺病毒载体。方法：PCR扩增含有CARP全长cDNA的片段，经测序验证无误后，亚克隆至pAdTrack-CMV穿梭质粒，再与pAdEasy．1质粒在大肠杆菌BJ5 183中进行同源重组产生腺病毒载体质粒。经过抗性筛选以及酶切鉴定得到阳性的重组质粒，再在293细胞中进行包装扩增，利用Adeasy系统上的绿色荧光蛋白标签鉴定病毒表达。结果：测序证实PCR产物为CARP全长cDNA；抗性筛选及酶切鉴定均表明重组腺病毒载体构建成功；转染293细胞3天后可见绿色荧光，回收病毒可以重复感染293细胞，证明病毒包装成功。     

Upregulated expression of cardiac ankyrin-repeated protein in renal podocytes is associated with proteinuria severity in lupus nephritis

Cardiac ankyrin-repeated protein (CARP) was originally identified as a protein specifically expressed in cardiomyocytes, but has recently been found to be upregulated in some muscle diseases including muscular dystrophy and myopathy, suggesting that CARP may be induced in some pathologic conditions. In this study, we immunohistochemically analyzed 69 renal biopsy samples from patients with glomerular diseases and 2 individuals with normal kidney. We found that CARP was expressed in renal podocytes at a high level in 10 of 13 cases of crescentic glomerulonephritis, 7 of 19 cases of diabetic nephropathy, and 12 of 20 cases of lupus nephritis, although it was not expressed in endocapillary glomerulonephritis, minimal change disease, thin basement membrane disease, membranous glomerulonephritis, and normal kidney. Interestingly, in lupus nephritis, CARP expression tended to be induced in cases exhibiting nephrotic syndrome, but less so in cases without nephrotic syndrome, suggesting that CARP expression is correlated with the severity of proteinuria. Furthermore, we found that CARP was not expressed in membranous glomerulonephritis but evidently expressed in most cases of membranous lupus nephritis. Although membranous glomerulonephritis and membranous lupus nephritis are sometimes morphologically indistinguishable, it is suggested that their pathologic mechanisms differ. Therefore, we propose that examination of CARP expression is useful for precise differential diagnosis of membranous glomerulonephritis and membranous lupus nephritis.     

Expression of cardiac ankyrin repeat protein, CARP, in malignant tumors: diagnostic use of CARP protein immunostaining in rhabdomyosarcoma

Cardiac ankyrin repeat protein (CARP) is highly expressed in cardiac muscles and detectable in normal skeletal muscles. Arpp, a close homolog of CARP, has been demonstrated to be useful for distinguishing rhabdomyosarcoma from other malignant tumors. However, the CARP distributions among malignant tumors have been poorly investigated. Here, we analyzed the comprehensive expression of CARP in malignant tumors and evaluated its potential use for rhabdomyosarcoma diagnosis. A total of 159 malignant tumors, including 34 rhabdomyosarcomas, 85 non-rhabdomyosarcomas, and 40 carcinomas, were immunohistochemically analyzed for CARP expression. Cytoplasmic expression of CARP was detected in 29 (85%) of 34 rhabdomyosarcomas. The immunoreactivity was observed in both small cells with little differentiation and differentiated tumor cells with abundant eosinophilic cytoplasm. In contrast, focal immunoreactivity for CARP was only observed in 5 (4%) of 125 non-rhabdomyosarcomas, comprising 2 malignant fibrous histiocytomas, 1 angiosarcoma, 1 epithelioid sarcoma, and 1 squamous cell carcinoma of the lung. Comparative analysis of the CARP expression profiles with those of myogenic markers in rhabdomyosarcomas revealed that myogenin (88%) and desmin (88%) exhibited the best sensitivity, followed by CARP (85%), MyoD (82%), muscle-specific actin (79%), and myoglobin (65%). MyoD (96%) and myoglobin (96%) had the best specificity, followed by CARP (95%), myogenin (95%), desmin (89%), and muscle-specific actin (86%). Our results indicate that CARP is a sensitive and specific marker for rhabdomyosarcoma and that it will be useful for the differential diagnosis of rhabdomyosarcoma.     

STAT3、MRP、LRP在肺腺癌A549及A549/CARP耐卡铂细胞株中的表达及关系的研究

目的:探明STAT3与肺腺癌细胞多药耐药之间的关系.方法:采用免疫组化法测定A549及A549/CARP耐卡铂细胞株中STAT3、LRP、MRP蛋白表达情况.结果:STAT3在A549组、A549/CARP组中阳性细胞数无显著性差异(t=1.519,P＞0.05);LRP、MRP在A549组、A549/CARP组中阳性细胞数有显著性差异(t=3.350,P＜0.01)(t=3.747,P＜0.01);STAT3、LRP、MRP之间表达均无相关性(r=0.061,P＞0.05).结论:STAT3并未直接参与介导肺腺癌的多药耐药性;LRP、MRP均参与了诱导肺腺癌的耐药,但二者之间无相关性;STAT3信号传导途径与LRP、MRP蛋白的激活机制可能无关.