miR-513a-5p慢病毒载体的构建及其对人骨肉瘤细胞放疗敏感性的影响

目的:构建miR-513a-5p慢病毒过表达载体，转染人骨肉瘤细胞株，观察miR-513a-5p对人骨肉瘤细胞放疗敏感性的影响。方法:PCR法扩增人miR-513a-5p基因，克隆入pLentis-CMV-GFP-MCS-PGK-PURO载体获得重组质粒pLentis-miR513a，双酶切鉴定并测序后将正确的重组质粒和对照质粒转染293FT细胞制备慢病毒，分别转染骨肉瘤HOS和U2OS细胞，qRT-PCR法及荧光显微镜鉴定转染结果。克隆形成实验、MTT法检测miR-513a-5p高表达HOS和U2OS细胞在X射线照射下细胞存活情况。结果:双酶切及测序结果确定成功构建miR-513a-5p慢病毒载体pLentis-miR513a。qRT-PCR结果提示，转染骨肉瘤细胞株后miR-513a-5p表达显著升高。克隆形成实验结果显示miR-513a-5p高表达后骨肉瘤细胞在X射线照射下细胞增殖减慢。MTT结果提示miR-513a-5p高表达骨肉瘤细胞经X射线照射后细胞存活减少。结论:成功构建了miR-513a-5p慢病毒载体，建立了高效稳定表达miR-513a-5p的骨肉瘤细胞株，高表达miR-513a-5p能显著增加X射线照射后骨肉瘤细胞的放疗敏感性。     

Intermittent Internal Fixation With a Locking Plate to Preserve Epiphyseal Growth Function During Limb-Salvage Surgery in a Child With Osteosarcoma of the Distal Femur: A Case Report

Limb shortening is a problem associated with surgery for osteosarcoma of the lower extremity in adolescents, as the tumors frequently occur near the epiphysis. Herein we report the use of a less invasive stabilization system (LISS) and an intermittent fixation method to preserve the growth function of epiphysis in an 11-year-old patient with an osteosarcoma of the distal femur.The 11-year-old male presented with left knee enlargement and pain for 2 weeks, and magnetic resonance imaging (MRI) and biopsy were consistent with osteosarcoma of the left distal femur. After preoperative chemotherapy, en bloc tumor resection was performed with margins based on MRI findings preserving the epiphyseal growth plate, the tumor cavity was filled with inactivated bone and bone cement, and a LISS was used to stabilize the femur. Aggressive postoperative chemotherapy was given. Approximately 105 weeks after surgery radiography showed that the distal end of the plate had moved superior to the epiphysis along with bone growth. Locking screws were placed in the distal part of the LISS plate to stabilize the re-implanted bone, and external fixation was not needed.The patient was able to walk with the crutches 1 week postoperatively, and bear weight on the extremity 6 weeks postoperatively. At 6 years after surgery, the patient''s height had increased 52 cm, shortening of the affected limb was only 1 cm, and the circumference of the affected limb was 2 cm smaller than that of the contralateral limb. There was no significant discomfort in the affected limb, and there was no gait abnormality. The patient could jump and run, and could participate in sports including basketball and badminton to the same degree as his peers.In summary, the novel method of bone reconstruction and fixation provided good results in a child with an osteosarcoma of the distal femur. This fixation method preserves the osteogenic function of the epiphysis and restored bone integrity simultaneously, and provides good functional recovery.     

Risk factors associated with delayed methotrexate clearance and increased toxicity in pediatric patients with osteosarcoma

High‐dose methotrexate (HD‐MTX; 12 g/m²) is part of standard therapy for pediatric osteosarcoma (OS). Risk factors associated with MTX toxicity in children with OS are not well defined. We investigated the association between peak MTX levels (four‐hour) and delayed MTX clearance or treatment toxicity. Information was retrieved from electronic medical records of 33 OS patients treated with HD‐MTX at Texas Children's Hospital from 2008 to 2015. We found that the four‐hour MTX level did not contribute to toxicity or delayed MTX clearance. We demonstrated that certain demographic characteristics are associated with delayed clearance and increased toxicity.     

人离体骨肉瘤组织血管噬菌体肽库筛选模型的建立及其意义

目的建立人离体骨肉瘤血管亲和筛选模型。方法(1)建立模型。术前对患肢局部行数字减影造影检查,标记肿瘤固有动脉大致走行,术后小心修剪,并连接类似于Langendorff的灌流装置,监控模型血管内的pH值、温度、氧分压等;(2)对噬菌体十二肽库进行筛选。结果28例骨肉瘤离体标本均建模成功,可直接用于灌流实验,并筛选出具有较高特异性的亲和短肽(基序为RLTR),各项指标显示离体骨肉瘤较好模拟了人体内的环境。结论建立人离体骨肉瘤血管亲和筛选模型是可行的,可直接用于对噬菌体随机肽库的亲和筛选,为骨肉瘤的靶向化疗提供参考。     

骨肉瘤与活化B淋巴细胞融合疫苗的抗肿瘤效应

目的　建立骨肉瘤与活化 B淋巴细胞融合的肿瘤疫苗 ,探讨其生物学与抗肿瘤特性 .方法　以 50 0 g· L-1 聚乙二醇为融合剂 ,将 C3 h小鼠来源的 HGRPT基因缺陷型 LM9骨肉瘤细胞与脂多糖活化的小鼠 B淋巴细胞进行融合 .经HAT选择性培养基筛选后 ,再观察该融合瘤株的体内外生长特性 ,对小鼠的致瘤性以及抗肿瘤活性 .结果　该融合瘤株体外生长缓慢 ,对 C3 h小鼠无致瘤性 ,杂交瘤细胞细胞表达B7(70 .6% ) H- 2 Kb (53.4% ) ,融合疫苗保护的小鼠可获 1 0 0 %无瘤生存 (8/ 8)而 LM9和射线照射后的 L M9对照组小鼠全部死亡 (0 / 8) ,皮下接种融合疫苗治疗的荷瘤小鼠可获得80 %无瘤生存 .结论　活化 B淋巴细胞融合骨肉瘤细胞可能改变其生物学特性 ,对小鼠有较好的预防和治疗作用 ,说明了细胞融合方法构建的肿瘤疫苗具有潜在的应用价值     

应用激光显微分离技术在骨肉瘤组织原位筛选噬菌体肽库

目的将激光显微分离(laser capture microdissection, LCM) 技术应用于噬菌体表面呈现肽库的筛选过程,建立一种可以直接在天然组织中筛选肽库的方法。方法将新鲜人骨肉瘤组织块在噬菌体肽库溶液振荡孵育后制成组织冰冻切片,免疫组化染色检测噬菌体在组织中的浸润扩散。改进常规LCM切片处理方法,以冻干法替代酒精/二甲苯法脱水,以期在LCM操作过程中提高切片上噬菌体的存活率。LCM法分离摄取骨肉瘤切片上的肿瘤靶细胞,转染回收这些细胞上特异结合的噬菌体。滴定法检测所筛选的噬菌体对特异性细胞的亲和力。结果利用LCM技术,可以由肿瘤冰冻切片上收集到足够的与特异性噬菌体短肽,应用于噬菌体表面呈现肽库筛选。经过3轮筛选后所获得的噬菌体与人骨肉瘤组织的特异性亲和力提高16倍。结论本研究首次将LCM技术应用于噬菌体表面呈现肽库的筛选,可以使我们直接在新鲜人肿瘤组织中筛选与特定细胞群甚至单个细胞亲合的短肽;同时又避免了天然组织中其他杂质细胞的污染,为研制细胞特异性导向载体提供了一个新的途径。     

Osteogenic Sarcoma of the Left Atrium

A primary cardiac osteogenic sarcoma is described in a patient presenting with a left atrial obstructive lesion. Wide surgical excision was possible with left atrial reconstruction and mitral valve replacement. The patient survived the operation and was symptom free for 1 year, finally dying at 18 months of cerebral metastases.     

骨肉瘤增殖细胞核抗原（PCNA）表达及其与临床病理和预后的关系

应用单克隆抗体ＰＣ１０免疫组方法半定量分析了增殖细胞核抗原表达与５０例骨肉瘤患者预后的关系。ＰＣＮＡ阳性级者预后差，其中半定量分级与骨肉瘤术后生存期呈显著负相关。ＰＣＮＡ表达与骨肉瘤组织学分级及骨样组织分化程度相关。     

保留骨骺灭活再植术治疗儿童股骨远端骨肉瘤

[目的]探讨保留骨骺灭活再植术的临床应用注意事项及并发症的防治。[方法]回顾6年间所治疗的11例患者,男5例,女6例。平均年龄7.6岁。均位于股骨下端。MRI分型:Ⅰ型7例,Ⅱ型4例。1例合并病理骨折。治疗方法均采用术前化疗2疗程 保留骨骺灭活再植手术 术后化疗。[结果]手术时间为3~4 h。术中出血量为300~500 ml。无血管、神经损伤。术后切口一期愈合,无切口感染及切口延迟愈合。11例获得随访10~72个月,患膝屈曲>110°3例,90°~110°3例,60°~89°4例,<60°1例。下肢等长4例,患肢较健侧短<2 cm 5例,2~3cm 2例。复发1例,转移2例,死亡3例,螺钉松动1例,灭活骨骨折1例。[结论]保留骨骺灭活再植术有利于术后功能恢复和肢体长度的保持;严格无瘤及无菌操作、彻底引流、适当延长外固定的时间、功能锻炼时注意保护肢体等是预防并发症发生的主要措施。     

Construction of Antisense Transforming Growth Factorβ_1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

Summary:To construct the antisense transforming growth factorβ1(TGFβ1)gene and investigatethe effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells.TGFβ1 cDNAwas cloned by RT-PCR from human osteosarcoma cells(MG-63)and inserted into pcDNA_3 to con-struct an antisense expression vector,which was dubbed pcDNA_3-TGFβ1(-).MTT was used to de-tect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene.Our results showedthat the proliferation of the transfected osteosarcoma cells was suppressed markedly.It is concludedthat TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation,which mightbe helpful for gene therapy of osteosarcoma.